U.S. patent application number 15/549962 was filed with the patent office on 2019-04-18 for enhanced delivery of viral particles to the striatum and cortex.
This patent application is currently assigned to Genzyme Corporation. The applicant listed for this patent is Genzyme Corporation. Invention is credited to Lamya SHIHABUDDIN, Lisa M. STANEK.
Application Number | 20190111157 15/549962 |
Document ID | / |
Family ID | 56614902 |
Filed Date | 2019-04-18 |
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United States Patent
Application |
20190111157 |
Kind Code |
A1 |
STANEK; Lisa M. ; et
al. |
April 18, 2019 |
ENHANCED DELIVERY OF VIRAL PARTICLES TO THE STRIATUM AND CORTEX
Abstract
Provided herein are novel methods for delivering recombinant
adeno-associated viral (rAAV) particles to the central nervous
system of a mammal (e.g., a human). In aspects, the methods involve
administering rAAV particles containing a heterologous nucleic acid
to the striatum and causing expression of the heterologous nucleic
acid in at least the cerebral cortex and the striatum of the
mammal.
Inventors: |
STANEK; Lisa M.; (Natick,
MA) ; SHIHABUDDIN; Lamya; (West Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genzyme Corporation |
Cambridge |
VA |
US |
|
|
Assignee: |
Genzyme Corporation
Cambridge
MA
Genzyme Corporation
Cambridge
MA
|
Family ID: |
56614902 |
Appl. No.: |
15/549962 |
Filed: |
February 9, 2016 |
PCT Filed: |
February 9, 2016 |
PCT NO: |
PCT/US16/17210 |
371 Date: |
August 9, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62114544 |
Feb 10, 2015 |
|
|
|
62220997 |
Sep 19, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2750/14122
20130101; C12N 7/00 20130101; C12N 2750/14152 20130101; A61P 25/00
20180101; A61P 25/08 20180101; C12N 15/86 20130101; A61K 9/0085
20130101; A61P 25/28 20180101; A61K 48/0075 20130101; C12N
2750/14143 20130101; A61P 25/14 20180101; A61P 25/16 20180101; A61P
43/00 20180101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 9/00 20060101 A61K009/00; C12N 7/00 20060101
C12N007/00 |
Claims
1. A method for delivering a recombinant adeno-associated viral
(rAAV) particle to the central nervous system of a mammal
comprising administering the rAAV particle to the striatum, wherein
the rAAV particle comprises an rAAV vector encoding a heterologous
nucleic acid that is expressed in at least the cerebral cortex and
striatum of the mammal.
2. The method of claim 1, wherein the rAAV particle comprises an
AAV serotype 1 (AAV1) capsid.
3. The method of claim 1, wherein the rAAV particle comprises an
AAV serotype 2 (AAV2) capsid.
4. (canceled)
5. The method of claim 1, wherein the rAAV particle is administered
to the putamen and the caudate nucleus of the striatum.
6. The method of claim 5, wherein the rAAV particle is administered
to the putamen and the caudate nucleus of each hemisphere of the
striatum.
7. The method of claim 5, wherein the rAAV particle is administered
to one site in the caudate nucleus and two sites in the
putamen.
8. The method of claim 5, wherein the ratio of rAAV particles
administered to the putamen to rAAV particles administered to the
caudate nucleus is at least about 2:1.
9. The method of claim 1, wherein the heterologous nucleic acid is
expressed in the frontal cortex, occipital cortex, and/or layer IV
of the mammal.
10. (canceled)
11. The method of claim 1, wherein the rAAV particle undergoes
retrograde or anterograde transport in the cerebral cortex.
12. The method of claim 1, wherein the heterologous nucleic acid is
further expressed in the thalamus, subthalamic nucleus, globus
pallidus, substantia nigra and/or hippocampus.
13-14. (canceled)
15. The method of claim 1, wherein the rAAV vector comprises the
heterologous nucleic acid flanked by one or more AAV inverted
terminal repeat (ITR) sequences.
16-18. (canceled)
19. The method of claim 15, wherein the ITR and the capsid of the
rAAV particle are derived from the same AAV serotype.
20. (canceled)
21. The method of claim 15, wherein the ITR and the capsid of the
rAAV viral particles are derived from different AAV serotypes.
22-30. (canceled)
31. The method of claim 1, wherein the rAAV vector is a
self-complementary rAAV vector.
32-33. (canceled)
34. The method of claim 1, wherein the heterologous nucleic acid
encodes a therapeutic polypeptide or therapeutic nucleic acid.
35-45. (canceled)
46. The method of claim 1, wherein the rAAV particle is delivered
by stereotactic delivery.
47. The method of claim 1, wherein the rAAV particle is delivered
by convection enhanced delivery.
48-53. (canceled)
54. A method of treating a disorder of the CNS in a mammal
comprising administering an effective amount of a rAAV particle to
the striatum of the mammal, wherein the rAAV particle comprises an
rAAV vector encoding a heterologous nucleic acid that is expressed
in at least the cerebral cortex and striatum of the mammal.
55. The method of claim 54, wherein the disorder of the CNS is
Huntington's Disease.
56. (canceled)
57. The method of claim 54, wherein the disorder of the CNS is
Parkinson's Disease.
58-89. (canceled)
90. The method of claim 55, wherein the heterologous nucleic acid
encodes a therapeutic polypeptide or a therapeutic nucleic acid
that inhibits the expression of HTT or inhibits the accumulation of
HTT in cells of the CNS of the mammal.
91. (canceled)
92. The method of claim 55, wherein the heterologous nucleic acid
encodes a miRNA that targets huntingtin.
93. (canceled)
94. The method of claim 57, wherein the heterologous nucleic acid
encodes a therapeutic polypeptide, wherein the therapeutic
polypeptide is glial-derived growth factor (GDNF), brain-derived
growth factor (BDNF), tyrosine hydroxlase (TH), GTP-cyclohydrolase
(GTPCH), and/or amino acid decarboxylase (AADC).
95-106. (canceled)
107. A system for expression of a heterologous nucleic acid in the
cerebral cortex and striatum of a mammal, comprising a) a
composition comprising rAAV particles, wherein the rAAV particles
comprise a rAAV vector encoding the heterologous nucleic acid; and
b) a device for delivery of the rAAV particles to the striatum.
108-156. (canceled)
157. A kit for delivering a rAAV particle to the CNS of a mammal or
for treating a disorder of the CNS in a mammal, comprising rAAV
particles, wherein the rAAV particles comprise a rAAV vector
encoding a heterologous nucleic acid that is expressed in at least
the cerebral cortex and striatum of the mammal.
158-222. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a National Phase application under 35
U.S.C. .sctn. 371 of International Application No.
PCT/US2016/017210 filed Feb. 9, 2016, which claims priority to U.S.
Provisional Application No. 62/114,544, filed Feb. 10, 2015, and
U.S. Provisional Application No. 62/220,997, filed Sep. 19, 2015,
each of which is incorporated herein by reference in its
entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
159792012700SeqList.txt, date recorded: Aug. 9, 2017, size: 1
KB).
FIELD OF THE INVENTION
[0003] The present invention relates to the delivery of AAV gene
therapy vectors to the brain, e.g., the striatum and/or cortex.
SUMMARY OF THE INVENTION
[0004] Adeno-associated virus (AAV)-based vectors have become the
preferred vector system for neurologic gene therapy, with an
excellent safety record established in multiple clinical trials
(Kaplitt et al., (2007) Lancet 369:2097-2105; Eberling et al.,
(2008) Neurology 70:1980-1983; Fiandaca et al., (2009) Neuroimage
47 Suppl. 2:T27-35). Effective treatment of neurologic disorders
has been hindered by problems associated with the delivery of AAV
vectors to affected cell populations. This delivery issue has been
especially problematic for disorders involving the cerebral cortex.
Simple injections do not distribute AAV vectors effectively,
relying on diffusion, which is effective only within a 1- to 3-mm
radius. An alternative method, convection-enhanced delivery (CED)
(Nguyen et al., (2003) J. Neurosurg. 98:584-590), has been used
clinically in gene therapy (AAV2-hAADC) for Parkinson's disease
(Fiandaca et al., (2008) Exp. Neurol. 209:51-57). The underlying
principle of CED involves pumping infusate into brain parenchyma
under sufficient pressure to overcome the hydrostatic pressure of
interstitial fluid, thereby forcing the infused particles into
close contact with the dense perivasculature of the brain.
Pulsation of these vessels acts as a pump, distributing the
particles over large distances throughout the parenchyma (Hadaczek
et al., (2006) Hum. Gene Ther. 17:291-302). To increase the safety
and efficacy of CED a reflux-resistant cannula (Krauze et al.,
(2009)Methods Enzymol. 465:349-362) can be employed along with
monitored delivery with real-time MRI. Monitored delivery allows
for the quantification and control of aberrant events, such as
cannula reflux and leakage of infusate into ventricles (Eberling et
al., (2008) Neurology 70:1980-1983; Fiandaca et al., (2009)
Neuroimage 47 Suppl. 2:T27-35; Saito et al., (2011) Journal of
Neurosurgery Pediatrics 7:522-526). However, there is still a need
for improved procedures to achieve widespread expression of AAV
vectors in the cortex and/or striatum.
[0005] The invention provides a method for delivering a recombinant
adeno-associated viral (rAAV) particle to the central nervous
system of a mammal comprising administering the rAAV particle to
the striatum, wherein the rAAV particle comprises a rAAV vector
encoding a heterologous nucleic acid that is expressed in at least
the cerebral cortex and striatum of the mammal. In some aspects,
the invention provides a method for delivering a rAAV particle to
the central nervous system of a mammal comprising administering the
rAAV particle to the striatum, wherein the rAAV particle comprises
an rAAV vector encoding a heterologous nucleic acid that is
expressed in at least the cerebral cortex and striatum of the
mammal and wherein the rAAV particle comprises an AAV serotype 1
(AAV1) capsid. In some aspects, the invention provides a method for
delivering a rAAV particle to the central nervous system of a
mammal comprising administering the rAAV particle to the striatum,
wherein the rAAV particle comprises an rAAV vector encoding a
heterologous nucleic acid that is expressed in at least the
cerebral cortex and striatum of the mammal and wherein the rAAV
particle comprises an AAV serotype 2 (AAV2) capsid. In some
embodiments, the mammal is a human.
[0006] In some embodiments, the rAAV particle is administered to at
least the putamen and the caudate nucleus of the striatum. In some
embodiments, the rAAV particle is administered to at least the
putamen and the caudate nucleus of each hemisphere of the striatum.
In some embodiments, the rAAV particle is administered to at least
one site in the caudate nucleus and two sites in the putamen. In
some embodiments, the ratio of rAAV particles administered to the
putamen to rAAV particles administered to the caudate nucleus is at
least about 2:1. In some embodiments, the heterologous nucleic acid
is expressed in at least the frontal cortex, occipital cortex,
and/or layer IV of the mammal. In some embodiments, the
heterologous nucleic acid is expressed at least in the prefrontal
association cortical areas, the premotor cortex, the primary
somatosensory cortical areas, sensory motor cortex, parietal
cortex, occipital cortex, and/or primary motor cortex. In some
embodiments, the rAAV particle undergoes retrograde or anterograde
transport in the cerebral cortex. In some embodiments, the
heterologous nucleic acid is further expressed in the thalamus,
subthalamic nucleus, globus pallidus, substantia nigra and/or
hippocampus. In some embodiments, the rAAV particle is administered
to the caudate nucleus and the putamen at a rate of greater than 1
.mu.L/min to about 5 .mu.L/min.
[0007] In some embodiments of the above aspects and embodiments,
the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12,
AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A,
AAV V708K, a goat AAV, AAV1/AAV2 chimeric, bovine AAV, or mouse AAV
capsid rAAV2/HBoV1 serotype capsid. In some embodiments, the AAV
serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R,
AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV vector
comprises the heterologous nucleic acid flanked by one or more AAV
inverted terminal repeat (ITR) sequences. In some embodiments, the
heterologous nucleic acid is flanked by two AAV ITRs. In some
embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12,
AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype
ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some
embodiments, the ITR and the capsid of the rAAV particle are
derived from the same AAV serotype. In some embodiments, the ITR
and the capsid are derived from AAV2. In other embodiments, the ITR
and the capsid of the rAAV viral particles are derived from
different AAV serotypes. In some embodiments, the ITR is derived
from AAV2 and the capsid is derived from AAV1.
[0008] In some embodiments of the above aspects and embodiments,
the heterologous nucleic acid is operably linked to a promoter. In
some embodiments, the promoter expresses the heterologous nucleic
acid in a cell of the CNS. In some embodiments, the promoter
expresses the heterologous nucleic acid in a brain cell. In some
embodiments, the promoter expresses the heterologous nucleic acid
in a neuron and/or a glial cell. In some embodiments, the neuron is
a medium spiny neuron of the caudate nucleus, a medium spiny neuron
of the putamen, a neuron of the cortex layer IV and/or a neuron of
the cortex layer V. In some embodiments, the glial cell is an
astrocyte. In some embodiments, the promoter is a CBA promoter, a
minimum CBA promoter, a CMV promoter or a GUSB promoter. In other
embodiments, the promoter is inducible. In further embodiments, the
rAAV vector comprises one or more of an enhancer, a splice
donor/splice acceptor pair, a matrix attachment site, or a
polyadenylation signal. In some embodiments, the rAAV vector is a
self-complementary rAAV vector. In some embodiments, the vector
comprises a first nucleic acid sequence encoding the heterologous
nucleic acid and a second nucleic acid sequence encoding a
complement of the heterologous nucleic acid, wherein the first
nucleic acid sequence can form intrastrand base pairs with the
second nucleic acid sequence along most or all of its length. In
some embodiments, the first nucleic acid sequence and the second
nucleic acid sequence are linked by a mutated AAV ITR, wherein the
mutated AAV ITR comprises a deletion of the D region and comprises
a mutation of the terminal resolution sequence.
[0009] In some embodiments of the above aspects and embodiments,
the heterologous nucleic acid encodes a therapeutic polypeptide or
therapeutic nucleic acid. In some embodiments, the heterologous
nucleic acid encodes a therapeutic polypeptide. In some
embodiments, the therapeutic polypeptide is an enzyme, a
neurotrophic factor, a polypeptide that is deficient or mutated in
an individual with a CNS-related disorder, an antioxidant, an
anti-apoptotic factor, an anti-angiogenic factor, and an
anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase
(GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS),
galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase
(MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA),
N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid
sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid
alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB,
N-sulfoglucosamine sulfohydrolase (MPS3A),
N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA,
alpha-glucosaminidase N-acetyltransferase (MPS3C),
N-acetylglucosamine-6-sulfatase (GNS),
alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB),
hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal
acid lipase (LIPA), Aspartylglucosaminidase, Alpha-galactosidase A,
Palmitoyl protein thioesterase, Tripeptidyl peptidase, Lysosomal
transmembrane protein, Cysteine transporter, Acid ceramidase, Acid
alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, Arylsulfatase
B, Arylsulfatase A, N-acetylgalactosamine-6-sulfate, Acid
beta-galactosidase, or alpha-neuramidase. In other embodiments, the
heterologous nucleic acid encodes a therapeutic nucleic acid. In
some embodiments, the therapeutic nucleic acid is an siRNA, an
shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a
DNAzyme. In some embodiments, the therapeutic polypeptide or the
therapeutic nucleic acid is used to treat a disorder of the
CNS.
[0010] In some embodiments of the above aspects and embodiments,
the disorder of the CNS is a lysosomal storage disease (LSD),
Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's
disease, stroke, corticobasal degeneration (CBD), corticogasal
ganglionic degeneration (CBGD), frontotemporal dementia (FTD),
multiple system atrophy (MSA), progressive supranuclear palsy (PSP)
or cancer of the brain. In some embodiments, the disorder is a
lysosomal storage disease selected from the group consisting of
Aspartylglusoaminuria, Fabry, Infantile Batten Disease (CNL1),
Classic Late Infantile Batten Disease (CNL2), Juvenile Batten
Disease (CNL3), Batten form CNL4, Batten form CNLS, Batten form
CNL6, Batten form CNL7, Batten form CNL8, Cystinosis, Farber,
Fucosidosis, Galactosidosialidosis, Gaucher disease type 1, Gaucher
disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter
disease, Krabbe disease, a mannosidosis disease, 13 mannosidosis
disease, Maroteaux-Lamy, metachromatic leukodystrophy disease,
Morquio A, Morquio B, mucolipidosisII/III disease, Niemann-Pick A
disease, Niemann-Pick B disease, Niemann-Pick C disease, Pompe
disease, Sandhoff disease, Sanfillipo A disease, Sanfillipo B
disease, Sanfillipo C disease, Sanfillipo D disease, Schindler
disease, Schindler-Kanzaki, sialidosis, Sly disease, Tay-Sachs
disease, and Wolman disease.
[0011] In some embodiments of the above aspects and embodiments,
the rAAV particle is in a composition. In further embodiments, the
composition is a pharmaceutical composition comprising a
pharmaceutically acceptable excipient.
[0012] In some embodiments of the above aspects and embodiments,
the rAAV particle was produced by triple transfection of a nucleic
acid encoding the rAAV vector, a nucleic acid encoding AAV rep and
cap, and a nucleic acid encoding AAV helper virus functions into a
host cell, wherein the transfection of the nucleic acids to the
host cells generates a host cell capable of producing rAAV
particles. In other embodiments, the rAAV particle was produced by
a producer cell line comprising one or more of nucleic acid
encoding the rAAV vector, a nucleic acid encoding AAV rep and cap,
and a nucleic acid encoding AAV helper virus functions.
[0013] In some embodiments of the above aspects and embodiments,
the rAAV particle is delivered by stereotactic delivery. In some
embodiments, the rAAV particle is delivered by convection enhanced
delivery. In some embodiments, the rAAV particle is delivered using
a CED delivery system. In some embodiments, the CED system
comprises a cannula. In some embodiments, the cannula is a
reflux-resistant cannula or a stepped cannula. In some embodiments,
the CED system comprises a pump. In some embodiments, the pump is a
manual pump. In some embodiments, the pump is an osmotic pump. In
some embodiments, the pump is an infusion pump.
[0014] In some aspects, the invention provides a method for
delivering rAAV particles to the central nervous system of a mammal
comprising administering a composition comprising the rAAV
particles to the striatum by CED, wherein the composition is
administered to the striatum at a rate of greater than 1 .mu.L/min
to about 5 .mu.L/min. In some aspects, the invention provides a
method for delivering rAAV particles to the central nervous system
of a mammal comprising administering a composition comprising the
rAAV particles to the striatum by CED, wherein the composition
comprises rAAV particles and poloxamer. In some embodiments, the
poloxamer is poloxamer 188. In some embodiments, the concentration
of poloxamer in the composition is ranges from about 0.0001% to
about 0.01%. In some embodiments, the concentration of poloxamer in
the composition is about 0.001%. In some embodiments, the
composition further comprises sodium chloride, wherein the
concentration of sodium chloride in the composition ranges from
about 100 mM to about 250 mM. In some embodiments, the
concentration of sodium chloride in the composition is about 180
mM. In some embodiments, the composition further comprises sodium
phosphate, wherein the concentration of sodium phosphate in the
composition ranges from about 5 mM to about 20 mM and the pH is
about 7.0 to about 8.0. In some embodiments, the composition
further comprises sodium phosphate, wherein the concentration of
sodium phosphate in the composition is about 10 mM and the pH is
about 7.5. In some embodiments, the composition is administered to
the caudate nucleus and the putamen at a rate of greater than 1
.mu.L/min to about 5 .mu.L/min. In some embodiments, the amount of
the composition delivered to the putamen is about twice the volume
delivered to the caudate nucleus. In some embodiments, about 20
.mu.L to about 50 .mu.L of the composition is administered to the
caudate nucleus of each hemisphere and about 40 .mu.L to about 100
.mu.L of the composition is administered to the putamen of each
hemisphere. In some embodiments, about 30 .mu.L of the composition
is administered to the caudate nucleus of each hemisphere and about
60 .mu.L of the composition is administered to the putamen of each
hemisphere.
[0015] In some embodiments, the invention provides a method of
treating a disorder of the CNS in a mammal comprising administering
an effective amount of a rAAV particle to the mammal by the methods
described above.
[0016] In some aspects, the invention provides a method of treating
Huntington's Disease in a mammal comprising administering an
effective amount of a rAAV particle to the striatum, wherein the
rAAV particle comprises an rAAV vector encoding a heterologous
nucleic acid that is expressed in at least the cerebral cortex and
striatum of the mammal. In some embodiments, the rAAV particle
comprises an AAV1 capsid or an AAV2 capsid. In other aspects, the
invention provides a method of treating Parkinson's disease in a
mammal comprising administering an effective amount of a rAAV
particle to the striatum, wherein the rAAV particle comprises a
rAAV vector encoding a heterologous nucleic acid that is expressed
in at least the cerebral cortex and striatum of the mammal. In some
embodiments, the rAAV particle comprises an AAV2 capsid. In some
embodiments, the mammal is a human.
[0017] In some embodiments, the rAAV particle is administered to at
least the putamen and the caudate nucleus of the striatum. In some
embodiments, the rAAV particle is administered to at least the
putamen and the caudate nucleus of each hemisphere of the striatum.
In some embodiments, the rAAV particle is administered to at least
one site in the caudate nucleus and two sites in the putamen. In
some embodiments, the ratio of rAAV particles administered to the
putamen to rAAV particles administered to the caudate nucleus is at
least about 2:1. In some embodiments, the heterologous nucleic acid
is expressed in at least the frontal cortex, occipital cortex,
and/or layer IV of the mammal. In some embodiments, the
heterologous nucleic acid is expressed at least in the prefrontal
association cortical areas, the premotor cortex, the primary
somatosensory cortical areas, sensory motor cortex, parietal
cortex, occipital cortex, and/or primary motor cortex. In some
embodiments, the rAAV particle undergoes retrograde or anterograde
transport in the cerebral cortex. In some embodiments, the
heterologous nucleic acid is further expressed in the thalamus,
subthalamic nucleus, globus pallidus, substantia nigra and/or
hippocampus.
[0018] In some embodiments of the above aspects and embodiments,
the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12,
AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A,
AAV V708K, a goat AAV, AAV1/AAV2 chimeric, bovine AAV, or mouse AAV
capsid rAAV2/HBoV1 serotype capsid. In some embodiments, the AAV
serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R,
AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV vector
comprises the heterologous nucleic acid flanked by one or more AAV
inverted terminal repeat (ITR) sequences. In some embodiments, the
heterologous nucleic acid is flanked by two AAV ITRs. In some
embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12,
AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype
ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some
embodiments, the ITR and the capsid of the rAAV particle are
derived from the same AAV serotype. In some embodiments, the ITR
and the capsid are derived from AAV2. In other embodiments, the ITR
and the capsid of the rAAV viral particles are derived from
different AAV serotypes. In some embodiments, the ITR is derived
from AAV2 and the capsid is derived from AAV1.
[0019] In some embodiments of the above aspects and embodiments,
the heterologous nucleic acid is operably linked to a promoter. In
some embodiments, the promoter expresses the heterologous nucleic
acid in a cell of the CNS. In some embodiments, the promoter
expresses the heterologous nucleic acid in a brain cell. In some
embodiments, the promoter expresses the heterologous nucleic acid
in a neuron and/or a glial cell. In some embodiments, the neuron is
a medium spiny neuron of the caudate nucleus, a medium spiny neuron
of the putamen, a neuron of the cortex layer IV and/or a neuron of
the cortex layer V. In some embodiments, the glial cell is an
astrocyte. In some embodiments, the promoter is a CBA promoter, a
minimum CBA promoter, a CMV promoter or a GUSB promoter. In other
embodiments, the promoter is inducible. In further embodiments, the
rAAV vector comprises one or more of an enhancer, a splice
donor/splice acceptor pair, a matrix attachment site, or a
polyadenylation signal. In some embodiments, the rAAV vector is a
self-complementary rAAV vector. In some embodiments, the vector
comprises a first nucleic acid sequence encoding the heterologous
nucleic acid and a second nucleic acid sequence encoding a
complement of the heterologous nucleic acid, wherein the first
nucleic acid sequence can form intrastrand base pairs with the
second nucleic acid sequence along most or all of its length. In
some embodiments, the first nucleic acid sequence and the second
nucleic acid sequence are linked by a mutated AAV ITR, wherein the
mutated AAV ITR comprises a deletion of the D region and comprises
a mutation of the terminal resolution sequence.
[0020] In some embodiments of the above aspects and embodiments,
the heterologous nucleic acid encodes a therapeutic polypeptide or
therapeutic nucleic acid. In some embodiments, the therapeutic
polypeptide or the therapeutic nucleic acid inhibits the expression
of HTT or inhibits the accumulation of HTT in cells of the CNS of
the mammal with Huntington's disease. In some embodiments, the
heterologous nucleic acid encodes an siRNA, an shRNA, an RNAi, an
miRNA, an antisense RNA, a ribozyme or a DNAzyme. In some
embodiments, the heterologous nucleic acid encodes a miRNA that
targets huntingtin. In some embodiments, the huntingtin comprises a
mutation associated with Huntington's disease.
[0021] In some embodiments of the above aspects and embodiments,
the heterologous nucleic acid encodes a therapeutic polypeptide or
therapeutic nucleic acid for treating Huntington's disease. In some
embodiments, the therapeutic polypeptide or the therapeutic nucleic
acid inhibits the expression of HTT or inhibits the accumulation of
HTT in cells of the CNS of the mammal with Huntington's disease. In
some embodiments, the heterologous nucleic acid encodes an siRNA,
an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a
DNAzyme. In some embodiments, the heterologous nucleic acid encodes
a miRNA that targets huntingtin. In some embodiments, the
huntingtin comprises a mutation associated with Huntington's
disease.
[0022] In some embodiments of the above aspects and embodiments,
the heterologous nucleic acid encodes a therapeutic polypeptide or
therapeutic nucleic acid for treating Parkinson's disease. In some
embodiments, the therapeutic polypeptide is glial-derived growth
factor (GDNF), brain-derived growth factor (BDNF), tyrosine
hydroxlase (TH), GTP-cyclohydrolase (GTPCH), and/or amino acid
decarboxylase (AADC).
[0023] In some embodiments of the above aspects and embodiments,
the rAAV particle is in a composition. In further embodiments, the
composition is a pharmaceutical composition comprising a
pharmaceutically acceptable excipient.
[0024] In some embodiments of the above aspects and embodiments,
the rAAV particle was produced by triple transfection of a nucleic
acid encoding the rAAV vector, a nucleic acid encoding AAV rep and
cap, and a nucleic acid encoding AAV helper virus functions into a
host cell, wherein the transfection of the nucleic acids to the
host cells generates a host cell capable of producing rAAV
particles. In other embodiments, the rAAV particle was produced by
a producer cell line comprising one or more of nucleic acid
encoding the rAAV vector, a nucleic acid encoding AAV rep and cap,
and a nucleic acid encoding AAV helper virus functions.
[0025] In some embodiments of the above aspects and embodiments,
the rAAV particle is delivered by stereotactic delivery. In some
embodiments, the rAAV particle is delivered by convection enhanced
delivery. In some embodiments, the rAAV particle is delivered using
a CED delivery system. In some embodiments, the CED system
comprises a cannula. In some embodiments, the cannula is a
reflux-resistant cannula or a stepped cannula. In some embodiments,
the CED system comprises a pump. In some embodiments, the pump is a
manual pump. In some embodiments, the pump is an osmotic pump. In
some embodiments, the pump is an infusion pump.
[0026] In some aspects, the invention provides a system for
expression of a heterologous nucleic acid in the cerebral cortex
and striatum of a mammal, comprising a) a composition comprising
rAAV particles, wherein the rAAV particles comprise a rAAV vector
encoding the heterologous nucleic acid; and b) a device for
delivery of the rAAV particles to the striatum. In some
embodiments, the rAAV particle comprises an AAV1 capsid or an AAV2
capsid. In some embodiments, the mammal is a human.
[0027] In some embodiments of the system of the invention, the rAAV
particle is administered to the putamen and the caudate nucleus of
the striatum. In some embodiments, the rAAV particle is
administered to at least one site in the caudate nucleus and two
sites in the putamen. In some embodiments, the ratio of rAAV
particles administered to the putamen to rAAV particles
administered to the caudate nucleus is at least about 2:1. In some
embodiments, the heterologous nucleic acid is expressed in at least
the frontal cortex, occipital cortex, and/or layer IV of the
mammal. In some embodiments, the heterologous nucleic acid is
expressed at least in the prefrontal association cortical areas,
the premotor cortex, the primary somatosensory cortical areas,
sensory motor cortex, parietal cortex, occipital cortex, and/or
primary motor cortex. In some embodiments, the rAAV particle
undergoes retrograde or anterograde transport in the cerebral
cortex. In some embodiments, the heterologous nucleic acid is
further expressed in the thalamus, subthalamic nucleus, globus
pallidus, substantia nigra and/or hippocampus.
[0028] In some embodiments of the system of the invention, the rAAV
particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,
AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12,
AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A,
AAV V708K, a goat AAV, AAV1/AAV2 chimeric, bovine AAV, or mouse AAV
capsid rAAV2/HBoV1 serotype capsid. In some embodiments, the AAV
serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R,
AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV vector
comprises the heterologous nucleic acid flanked by one or more AAV
inverted terminal repeat (ITR) sequences. In some embodiments, the
heterologous nucleic acid is flanked by two AAV ITRs. In some
embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12,
AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype
ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some
embodiments, the ITR and the capsid of the rAAV particle are
derived from the same AAV serotype. In some embodiments, the ITR
and the capsid are derived from AAV2. In other embodiments, the ITR
and the capsid of the rAAV viral particles are derived from
different AAV serotypes. In some embodiments, the ITR is derived
from AAV2 and the capsid is derived from AAV1.
[0029] In some embodiments of the system of the invention, the
heterologous nucleic acid is operably linked to a promoter. In some
embodiments, the promoter expresses the heterologous nucleic acid
in a cell of the CNS. In some embodiments, the promoter expresses
the heterologous nucleic acid in a brain cell. In some embodiments,
the promoter expresses the heterologous nucleic acid in a neuron
and/or a glial cell. In some embodiments, the neuron is a medium
spiny neuron of the caudate nucleus, a medium spiny neuron of the
putamen, a neuron of the cortex layer IV and/or a neuron of the
cortex layer V. In some embodiments, the glial cell is an
astrocyte. In some embodiments, the promoter is a CBA promoter, a
minimum CBA promoter, a CMV promoter or a GUSB promoter. In other
embodiments, the promoter is inducible. In further embodiments, the
rAAV vector comprises one or more of an enhancer, a splice
donor/splice acceptor pair, a matrix attachment site, or a
polyadenylation signal. In some embodiments, the rAAV vector is a
self-complementary rAAV vector. In some embodiments, the vector
comprises a first nucleic acid sequence encoding the heterologous
nucleic acid and a second nucleic acid sequence encoding a
complement of the heterologous nucleic acid, wherein the first
nucleic acid sequence can form intrastrand base pairs with the
second nucleic acid sequence along most or all of its length. In
some embodiments, the first nucleic acid sequence and the second
nucleic acid sequence are linked by a mutated AAV ITR, wherein the
mutated AAV ITR comprises a deletion of the D region and comprises
a mutation of the terminal resolution sequence.
[0030] In some embodiments of the system of the invention, the
heterologous nucleic acid encodes a therapeutic polypeptide or
therapeutic nucleic acid. In some embodiments, the heterologous
nucleic acid encodes a therapeutic polypeptide. In some
embodiments, the therapeutic polypeptide is an enzyme, a
neurotrophic factor, a polypeptide that is deficient or mutated in
an individual with a CNS-related disorder, an antioxidant, an
anti-apoptotic factor, an anti-angiogenic factor, and an
anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase
(GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS),
galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase
(MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA),
N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid
sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid
alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB,
N-sulfoglucosamine sulfohydrolase (MPS3A),
N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA,
alpha-glucosaminidase N-acetyltransferase (MPS3C),
N-acetylglucosamine-6-sulfatase (GNS),
alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB),
hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal
acid lipase (LIPA), Aspartylglucosaminidase, Alpha-galactosidase A,
Palmitoyl protein thioesterase, Tripeptidyl peptidase, Lysosomal
transmembrane protein, Cysteine transporter, Acid ceramidase, Acid
alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, Arylsulfatase
B, Arylsulfatase A, N-acetylgalactosamine-6-sulfate, Acid
beta-galactosidase, or alpha-neuramidase. In other embodiments, the
heterologous nucleic acid encodes a therapeutic nucleic acid. In
some embodiments, the therapeutic nucleic acid is an siRNA, an
shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a
DNAzyme. In some embodiments, the therapeutic polypeptide or the
therapeutic nucleic acid is used to treat a disorder of the
CNS.
[0031] In some embodiments of the system of the invention, the
disorder of the CNS is a lysosomal storage disease (LSD),
Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's
disease, stroke, corticobasal degeneration (CBD), corticogasal
ganglionic degeneration (CBGD), frontotemporal dementia (FTD),
multiple system atrophy (MSA), progressive supranuclear palsy (PSP)
or cancer of the brain. In some embodiments, the disorder is a
lysosomal storage disease selected from the group consisting of
Aspartylglusoaminuria, Fabry, Infantile Batten Disease (CNL1),
Classic Late Infantile Batten Disease (CNL2), Juvenile Batten
Disease (CNL3), Batten form CNL4, Batten form CNLS, Batten form
CNL6, Batten form CNL7, Batten form CNL8, Cystinosis, Farber,
Fucosidosis, Galactosidosialidosis, Gaucher disease type 1, Gaucher
disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter
disease, Krabbe disease, a mannosidosis disease, .beta.
mannosidosis disease, Maroteaux-Lamy, metachromatic leukodystrophy
disease, Morquio A, Morquio B, mucolipidosisII/III disease,
Niemann-Pick A disease, Niemann-Pick B disease, Niemann-Pick C
disease, Pompe disease, Sandhoff disease, Sanfillipo A disease,
Sanfillipo B disease, Sanfillipo C disease, Sanfillipo D disease,
Schindler disease, Schindler-Kanzaki, sialidosis, Sly disease,
Tay-Sachs disease, and Wolman disease.
[0032] In some embodiments, the rAAV of the invention comprises a
heterologous nucleic acid encoding a therapeutic polypeptide or
therapeutic nucleic acid for treating Huntington's disease. In some
embodiments, the therapeutic polypeptide or the therapeutic nucleic
acid inhibits the expression of HTT or inhibits the accumulation of
HTT in cells of the CNS of the mammal with Huntington's disease. In
some embodiments, the heterologous nucleic acid encodes an siRNA,
an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a
DNAzyme. In some embodiments, the heterologous nucleic acid encodes
a miRNA that targets huntingtin. In some embodiments, the
huntingtin comprises a mutation associated with Huntington's
disease.
[0033] In some embodiments, the rAAV particle of the invention
comprises a heterologous nucleic acid encodes a therapeutic
polypeptide or therapeutic nucleic acid for treating Parkinson's
disease. In some embodiments, the therapeutic polypeptide is
glial-derived growth factor (GDNF), brain-derived growth factor
(BDNF), tyrosine hydroxlase (TH), GTP-cyclohydrolase (GTPCH),
and/or amino acid decarboxylase (AADC).
[0034] In some embodiments of the system of the invention, the rAAV
particle is in a composition. In further embodiments, the
composition is a pharmaceutical composition comprising a
pharmaceutically acceptable excipient.
[0035] In some embodiments of the system of the invention, the rAAV
particle was produced by triple transfection of a nucleic acid
encoding the rAAV vector, a nucleic acid encoding AAV rep and cap,
and a nucleic acid encoding AAV helper virus functions into a host
cell, wherein the transfection of the nucleic acids to the host
cells generates a host cell capable of producing rAAV particles. In
other embodiments, the rAAV particle was produced by a producer
cell line comprising one or more of nucleic acid encoding the rAAV
vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid
encoding AAV helper virus functions.
[0036] In some embodiments of the system of the invention, the rAAV
particle is delivered by stereotactic delivery. In some
embodiments, the rAAV particle is delivered by convection enhanced
delivery. In some embodiments, the rAAV particle is delivered using
a CED delivery system. In some embodiments, the CED system
comprises a cannula. In some embodiments, the cannula is a
reflux-resistant cannula or a stepped cannula. In some embodiments,
the CED system comprises a pump. In some embodiments, the pump is a
manual pump. In some embodiments, the pump is an osmotic pump. In
some embodiments, the pump is an infusion pump.
[0037] In some aspects, the invention provides a kit for use in any
of the methods described above where the kit comprising rAAV
particles, wherein the rAAV particles comprise a rAAV vector
encoding a heterologous nucleic acid that is expressed in at least
the cerebral cortex and striatum of the mammal. In some
embodiments, the rAAV particles comprise an AAV serotype 1 (AAV1)
capsid. In some embodiments, the rAAV particles comprise an AAV
serotype 2 (AAV2) capsid.
[0038] In some aspects, the invention provides a kit for treating
Huntington's Disease in a mammal, comprising a composition
comprising an effective amount of rAAV particles, wherein the rAAV
particles comprise an rAAV vector encoding a heterologous nucleic
acid that is expressed in at least the cerebral cortex and striatum
of the mammal. In some aspects, the invention provides a kit for
treating Parkinson's disease in a mammal, comprising a composition
comprising an effective amount of rAAV particles, wherein the rAAV
particles comprise a rAAV vector encoding a heterologous nucleic
acid that is expressed in at least the cerebral cortex and striatum
of the mammal. In some embodiments, the rAAV particles of the kits
comprise an AAV serotype 1 (AAV1) capsid or an AAV serotype 2
(AAV2) capsid. In some embodiments, the kit further comprising a
device for delivery of the rAAV particles to the striatum. In some
embodiments, the rAAV particles of the kit are in a composition. In
some embodiments, the composition comprises a buffer and/or a
pharmaceutically acceptable excipient. In further embodiments, the
kit comprises instructions for delivery of the composition of rAAV
particles to the striatum.
[0039] In some aspects, the invention provides a rAAV particle for
use in any of the methods described above. In some aspects, the
invention provides a rAAV particle for use in delivering a
recombinant adeno-associated viral (rAAV) particle to the central
nervous system of a mammal, wherein the rAAV particle comprises a
rAAV vector encoding a heterologous nucleic acid that is expressed
in at least the cerebral cortex and striatum of the mammal. In some
aspects, the invention provides a rAAV particle for use in
delivering a recombinant adeno-associated viral (rAAV) particle to
the central nervous system of a mammal, wherein the rAAV particle
comprises a rAAV vector encoding a heterologous nucleic acid that
is expressed in at least the cerebral cortex and striatum of the
mammal, and wherein the rAAV particle further comprises an AAV
serotype 1 (AAV1) capsid. In some aspects, the invention provide a
rAAV particle for use in delivering a recombinant adeno-associated
viral (rAAV) particle to the central nervous system of a mammal,
wherein the rAAV particle comprises a rAAV vector encoding a
heterologous nucleic acid that is expressed in at least the
cerebral cortex and striatum of the mammal, and wherein the rAAV
particle further comprises an AAV serotype 1 (AAV2) capsid.
[0040] In some aspects, the invention provides a rAAV particle for
use in treating Huntington's disease in a mammal, wherein the rAAV
particle comprises a rAAV vector encoding a heterologous nucleic
acid that is expressed in at least the cerebral cortex and striatum
of the mammal. In some aspects, the invention provides a rAAV
particle for use in treating Parkinson's disease in a mammal,
wherein the rAAV particle comprises a rAAV vector encoding a
heterologous nucleic acid that is expressed in at least the
cerebral cortex and striatum of the mammal. In some embodiments,
the rAAV particle comprises an AAV2 capsid. In some embodiments,
the mammal is a human.
[0041] In some embodiments, the rAAV particle of the invention is
administered to at least the putamen and the caudate nucleus of the
striatum. In some embodiments, the rAAV particle is administered to
at least the putamen and the caudate nucleus of each hemisphere of
the striatum. In some embodiments, the rAAV particle is
administered to at least one site in the caudate nucleus and two
sites in the putamen. In some embodiments, the ratio of rAAV
particles administered to the putamen to rAAV particles
administered to the caudate nucleus is at least about 2:1. In some
embodiments, the heterologous nucleic acid is expressed in at least
the frontal cortex, occipital cortex, and/or layer IV of the
mammal. In some embodiments, the heterologous nucleic acid is
expressed at least in the prefrontal association cortical areas,
the premotor cortex, the primary somatosensory cortical areas,
sensory motor cortex, parietal cortex, occipital cortex, and/or
primary motor cortex. In some embodiments, the rAAV particle
undergoes retrograde or anterograde transport in the cerebral
cortex. In some embodiments, the heterologous nucleic acid is
further expressed in the thalamus, subthalamic nucleus, globus
pallidus, substantia nigra and/or hippocampus.
[0042] In some embodiments of the above aspects and embodiments,
the rAAV particle of the invention comprises an AAV1, AAV2, AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10,
AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A,
AAV2 E548A, AAV2 N708A, AAV V708K, a goat AAV, AAV1/AAV2 chimeric,
bovine AAV, or mouse AAV capsid rAAV2/HBoV1 serotype capsid. In
some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7,
AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some
embodiments, the rAAV vector comprises the heterologous nucleic
acid flanked by one or more AAV inverted terminal repeat (ITR)
sequences. In some embodiments, the heterologous nucleic acid is
flanked by two AAV ITRs. In some embodiments, the AAV ITRs are
AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R,
AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV,
bovine AAV, or mouse AAV serotype ITRs. In some embodiments, the
AAV ITRs are AAV2 ITRs. In some embodiments, the ITR and the capsid
of the rAAV particle are derived from the same AAV serotype. In
some embodiments, the ITR and the capsid are derived from AAV2. In
other embodiments, the ITR and the capsid of the rAAV viral
particles are derived from different AAV serotypes. In some
embodiments, the ITR is derived from AAV2 and the capsid is derived
from AAV1.
[0043] In some embodiments of the above aspects and embodiments,
the heterologous nucleic acid is operably linked to a promoter. In
some embodiments, the promoter expresses the heterologous nucleic
acid in a cell of the CNS. In some embodiments, the promoter
expresses the heterologous nucleic acid in a brain cell. In some
embodiments, the promoter expresses the heterologous nucleic acid
in a neuron and/or a glial cell. In some embodiments, the neuron is
a medium spiny neuron of the caudate nucleus, a medium spiny neuron
of the putamen, a neuron of the cortex layer IV and/or a neuron of
the cortex layer V. In some embodiments, the glial cell is an
astrocyte. In some embodiments, the promoter is a CBA promoter, a
minimum CBA promoter, a CMV promoter or a GUSB promoter. In other
embodiments, the promoter is inducible. In further embodiments, the
rAAV vector comprises one or more of an enhancer, a splice
donor/splice acceptor pair, a matrix attachment site, or a
polyadenylation signal. In some embodiments, the rAAV vector is a
self-complementary rAAV vector. In some embodiments, the vector
comprises a first nucleic acid sequence encoding the heterologous
nucleic acid and a second nucleic acid sequence encoding a
complement of the heterologous nucleic acid, wherein the first
nucleic acid sequence can form intrastrand base pairs with the
second nucleic acid sequence along most or all of its length. In
some embodiments, the first nucleic acid sequence and the second
nucleic acid sequence are linked by a mutated AAV ITR, wherein the
mutated AAV ITR comprises a deletion of the D region and comprises
a mutation of the terminal resolution sequence.
[0044] In some embodiments of the above aspects and embodiments,
the heterologous nucleic acid encodes a therapeutic polypeptide or
therapeutic nucleic acid. In some embodiments, the heterologous
nucleic acid encodes a therapeutic polypeptide. In some
embodiments, the therapeutic polypeptide is an enzyme, a
neurotrophic factor, a polypeptide that is deficient or mutated in
an individual with a CNS-related disorder, an antioxidant, an
anti-apoptotic factor, an anti-angiogenic factor, and an
anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase
(GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS),
galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase
(MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA),
N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid
sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid
alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB,
N-sulfoglucosamine sulfohydrolase (MPS3A),
N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA,
alpha-glucosaminidase N-acetyltransferase (MPS3C),
N-acetylglucosamine-6-sulfatase (GNS),
alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB),
hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal
acid lipase (LIPA), Aspartylglucosaminidase, Alpha-galactosidase A,
Palmitoyl protein thioesterase, Tripeptidyl peptidase, Lysosomal
transmembrane protein, Cysteine transporter, Acid ceramidase, Acid
alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, Arylsulfatase
B, Arylsulfatase A, N-acetylgalactosamine-6-sulfate, Acid
beta-galactosidase, or alpha-neuramidase. In other embodiments, the
heterologous nucleic acid encodes a therapeutic nucleic acid. In
some embodiments, the therapeutic nucleic acid is an siRNA, an
shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a
DNAzyme. In some embodiments, the therapeutic polypeptide or the
therapeutic nucleic acid is used to treat a disorder of the
CNS.
[0045] In some embodiments of the above aspects and embodiments,
the disorder of the CNS is a lysosomal storage disease (LSD),
Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's
disease, stroke, corticobasal degeneration (CBD), corticogasal
ganglionic degeneration (CBGD), frontotemporal dementia (FTD),
multiple system atrophy (MSA), progressive supranuclear palsy (PSP)
or cancer of the brain. In some embodiments, the disorder is a
lysosomal storage disease selected from the group consisting of
Aspartylglusoaminuria, Fabry, Infantile Batten Disease (CNL1),
Classic Late Infantile Batten Disease (CNL2), Juvenile Batten
Disease (CNL3), Batten form CNL4, Batten form CNLS, Batten form
CNL6, Batten form CNL7, Batten form CNL8, Cystinosis, Farber,
Fucosidosis, Galactosidosialidosis, Gaucher disease type 1, Gaucher
disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter
disease, Krabbe disease, a mannosidosis disease, .beta.
mannosidosis disease, Maroteaux-Lamy, metachromatic leukodystrophy
disease, Morquio A, Morquio B, mucolipidosisII/III disease,
Niemann-Pick A disease, Niemann-Pick B disease, Niemann-Pick C
disease, Pompe disease, Sandhoff disease, Sanfillipo A disease,
Sanfillipo B disease, Sanfillipo C disease, Sanfillipo D disease,
Schindler disease, Schindler-Kanzaki, sialidosis, Sly disease,
Tay-Sachs disease, and Wolman disease.
[0046] In some embodiments of the above aspects and embodiments,
the rAAV particle is in a composition. In further embodiments, the
composition is a pharmaceutical composition comprising a
pharmaceutically acceptable excipient.
[0047] In some embodiments of the above aspects and embodiments,
the rAAV particle was produced by triple transfection of a nucleic
acid encoding the rAAV vector, a nucleic acid encoding AAV rep and
cap, and a nucleic acid encoding AAV helper virus functions into a
host cell, wherein the transfection of the nucleic acids to the
host cells generates a host cell capable of producing rAAV
particles. In other embodiments, the rAAV particle was produced by
a producer cell line comprising one or more of nucleic acid
encoding the rAAV vector, a nucleic acid encoding AAV rep and cap,
and a nucleic acid encoding AAV helper virus functions.
[0048] In some embodiments of the above aspects and embodiments,
the rAAV particle is delivered by stereotactic delivery. In some
embodiments, the rAAV particle is delivered by convection enhanced
delivery. In some embodiments, the rAAV particle is delivered using
a CED delivery system. In some embodiments, the CED system
comprises a cannula. In some embodiments, the cannula is a
reflux-resistant cannula or a stepped cannula. In some embodiments,
the CED system comprises a pump. In some embodiments, the pump is a
manual pump. In some embodiments, the pump is an osmotic pump. In
some embodiments, the pump is an infusion pump.
[0049] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1 shows Rhesus monkey body weights, taken immediately
prior to surgery (black) and at the time of necropsy (gray), in
animals administered AAV1 and AAV2 vectors made by triple
transfection (TT) and producer cell line (PCL) processes.
[0051] FIGS. 2A-2D show representative brain sections stained for
GFP 30 days after infusion of AAV1-GFP (TT) into Rhesus monkey
caudate and putamen. Sections in FIGS. 2A-2D extend through the
brain in the rostral to caudal direction. Sections from three
representative animals are displayed in each panel.
[0052] FIGS. 3A-3D show representative brain sections demonstrating
cortical expression of GFP in the frontal cortex (FIGS. 3A &
3B) and occipital cortex (FIGS. 3C & 3D) in both astrocytes
(FIGS. 3A & 3C) and cortical neurons (FIGS. 3B & 3D) after
infusion of AAV1-GFP (TT) into Rhesus monkey caudate and
putamen.
[0053] FIGS. 4A-4D show representative brain sections stained for
GFP 30 days after infusion of AAV2-GFP (TT) into Rhesus monkey
caudate and putamen. Sections in FIGS. 4A-4D extend through the
brain in the rostral to caudal direction. Sections from three
representative animals are displayed in each panel.
[0054] FIGS. 5A-5D show representative brain sections stained for
GFP 30 days after infusion of AAV1-GFP made by producer cell lines
(PCL) (FIGS. 5A & 5B) or triple transfection (TT) (FIGS. 5C
& 5D) processes into Rhesus monkey caudate and putamen.
[0055] FIGS. 6A-6D show representative brain sections stained for
GFP 30 days after infusion of AAV2-GFP made by producer cell line
(PCL) (FIGS. 6A & 6B) or triple transfection (TT) (FIGS. 6C
& 6D) processes into Rhesus monkey caudate and putamen.
[0056] FIG. 7 shows the distribution of GFP in non-human primate
(NHP) brains infused with AAV1-eGFP and AAV2-eGFP. AAV1-eGFP and
AAV2-eGFP vectors were infused bilaterally into the striatum of 9
Rhesus macaques. Four weeks after the surgery, brains were
processed for immunohistochemistry (IHC) against GFP. Columns show
representative GFP-stained brain sections from 4 study groups
infused with AAV1-eGFP (Triple Transfection; TT); AAV1-eGFP
(Producer Cell Line; PCL); AAV2-eGFP (TT); AAV2-eGFP (PCL).
Representative sections show various coronal planes of the brain to
demonstrate distribution of GFP expression throughout the entire
brain from frontal cortex, striatum (infusion sites), midbrain, to
occipital parts of the cortex. All groups showed robust GFP signal
in the sites of injection (putamen and caudate nucleus) as well as
extensive transport to cortical regions and substantia nigra. Based
on the IHC staining, the coverage of GFP expression in both target
structure (striatum) and cortical regions were calculated for each
monkey and are summarized in Table 7.
[0057] FIG. 8 shows the ratios of primary areas of transduction
(PAT) to vector distribution (Vd). Primary areas of GFP expression
in the striatum were delineated on scans from the GFP-stained
sections and their values divided by values obtained from matching
MRI scans with Gadolinium signal. Ratios >1.0 indicate that the
extent of GFP expression exceeds the boundaries of Gadolinium
signal after infusion. The results from monkeys infused with AAV
vectors showed that AAV1 spreads better in the brain parenchyma
than AAV2 (1.21.+-.0.1 vs. 0.74.+-.0.04; p<0.007 with 2-tailed
unpaired t-test).
[0058] FIGS. 9A-9H show GFP expression in the NHP brain transduced
with AAV1-eGFP and AAV2-eGFP. FIG. 9A: High magnification
(40.times.) of the target structure caudate nucleus transduced with
AAV1-eGFP (TT) of subject number 1. Dark-brown GFP+ neurons stained
by DAB are visible against densely stained network of positive
neuronal fibers. Such a robust signal was detected in all monkeys
injected with AAV1-eGFP vector produced by both TT and PCL methods.
FIG. 9B: Fragment of prefrontal cortex of subject number 1 (FIG. 7)
demonstrating massive transport of vector AAV1-eGFP from the sites
of injection (striatum) to cortical regions. Based on morphology of
GFP+ cells, both neurons and astrocytes were detected in the
cortex. FIG. 9C: Higher magnification (40.times.) of the frame
indicated in FIG. 9B showing numerous cortical neurons expressing
GFP. FIG. 9D: High (40.times.) magnification of the cortex from
subject number 1 showing GFP+ cells of astrocytic morphology. FIG.
9E: High magnification (40.times.) of the target structure putamen
transduced with AAV2-eGFP (TT) of subject number 6. Dark-brown DAB
signal show expression of GFP in neurons and their densely stained
network of fibers. FIG. 9F: Fragment of prefrontal cortex of
subject number 6 (FIG. 7) demonstrating massive transport of vector
AAV2-eGFP from the striatum (injection site) to cortical regions.
The vast majority of GFP-positive cells had neuronal morphology
(magnification 2.5.times.). FIG. 9G: Higher magnification
(40.times.) of the frame indicated in FIG. 9F showing numerous
cortical neurons expressing GFP. FIG. 9H: Higher magnification
(20.times.) of internal capsule of subject number 6 showing GFP+
cells with astrocytic morphology.
[0059] FIGS. 10A-10E show the cellular tropism of AAV1-eGFP and
AAV2-eGFP injected into the monkey brain. Monkey brain sections
were processed for double immunofluorescence staining against GFP
and various cellular markers to determine cellular tropism of the
injected vectors. FIG. 10A: Section from caudate nucleus (target
structure) from subject number 1 stained with antibodies against
GFP (green channel for DyLight.TM. 488 dye; left column) and
neuronal marker NeuN (red channel for DyLight.TM. 549 dye; middle
column). Merged pictures (magnification 20.times.; right column)
from both channels show numerous neurons expressing GFP, verifying
neuronal tropism of AAV1-eGFP. FIG. 10B: The same staining was
performed for a section from prefrontal cortex of subject number 1
showing neuronal transduction in a distal brain structure receiving
neuronal projections from the striatum and is evidence of
retrograde transport of AAV1-eGFP. FIG. 10C: Section from caudate
nucleus (target structure) from subject number 1 stained with
antibodies against GFP (green channel for DyLight.TM. 488 dye; left
column) and astrocytic marker S-100 (red channel for DyLight.TM.
549 dye; middle column). Merged pictures (magnification 20.times.;
right column) from both channels show numerous astrocytes
expressing GFP, verifying that AAV1-eGFP also transduces
astrocytes. FIG. 10D: Section from caudate nucleus (target
structure) from subject number 6 stained with antibodies against
GFP (green channel for DyLight.TM. 488 dye; left column) and
neuronal marker NeuN (red channel for DyLight.TM. 549 dye; middle
column). Merged pictures (magnification 20.times.; right column)
from both channels show numerous neurons expressing GFP, verifying
neuronal tropism of AAV2-eGFP. FIG. 10E: Section from caudate
nucleus (target structure) from subject number 3 stained with
antibodies against GFP (green channel for DyLight.TM. 488 dye; left
column) and microglia marker Iba-1 (red channel for DyLight.TM. 549
dye; middle column). The lack of co-staining of both markers in
merged picture (magnification 20.times.; right column) indicates
that AAV1 does not transduce microglia, and this was also the case
for AAV2 (data not shown).
[0060] FIGS. 11A-11C show the efficiency of neuronal transduction
in the striatum of NHP injected with AAV1-eGFP and AAV2-eGFP.
Double immunofluorescence staining against GFP and neuronal marker
NeuN of monkey brain sections was performed to calculate the
efficiency of neuronal transduction within the striatum (target
structure) and cortical regions. For the striatum, the efficiency
of transduction was calculated in the primary area of GFP
transduction (PAT) where signal was robust with densely distributed
GFP+ neurons (FIG. 11A). Neurons were also detected in regions
outside the primary areas of GFP transduction (OPAT; FIG. 11C).
Scheme for the technique of counting GFP+ neurons in PAT (inner
shading) and OPAT (outer shading) is shown in FIG. 11B. Data from
individual counts for each monkey and brain structure are shown in
Table 8 (PAT) and Table 9 (OPAT).
[0061] FIGS. 12A & 12B show vector-related histological
findings. Independent evaluation of hematoxylin and eosin (H&E)
staining of coronal sections from areas of primary transduction
(PAT) revealed normal gliosis related to cannula insertion in all
experimental groups. H&E staining also revealed perivascular
cellular infiltrates in all animals regardless of the vector used.
The incidence and severity of perivascular cuffs was increased in
groups injected with AAV1, especially when the vector was prepared
by the TT method. FIG. 12A: H&E-stained section from subject
number 3 shows numerous perivascular cuffs in the left putamen
transduced with AAV1-eGFP (TT). One blood vessel is magnified
(5.times.) in the right bottom corner. FIG. 12B: H&E-stained
section from subject number 5 shows only a few localized
perivascular cuffs in the left caudate nucleus transduced with
AAV1-eGFP (PCL). A few blood vessels are magnified (5.times.) in
the left bottom corner.
[0062] FIGS. 13A & 13B show quantitative PCT (QPCR) analysis of
eGFP mRNA expression in liver, spleen, heart, kidney, and lung
samples 1 month following injection of AAV1-eGFP into Rhesus monkey
caudate and putamen. (FIG. 13A) AAV1 and AAV2-eGFP vectors made by
a triple transfection (TT) process. (FIG. 13B) AAV1 and AAV2-eGFP
vectors made by a producer cell line (PCL) process.
DETAILED DESCRIPTION
[0063] As discussed in detail herein, the inventors have discovered
that AAV vectors (e.g., AAV1 and AAV2 vectors) efficiently target
both striatal and cortical structures in the Rhesus monkey brain
when delivered to the striatum (e.g., by convection enhanced
delivery, CED). These studies also evaluated two different
manufacturing platforms, and these studies demonstrate that AAV
generated by triple transfection and producer cell lines target
both striatal and cortical structures in the Rhesus monkey brain.
Intrastriatal delivery of rAAV particles (e.g., AAV1 and AAV2
vectors) produced using both platforms was able to transduce
neurons located a considerable distance from the infusion site
(e.g., cortical structures), as well as neurons in the striatum.
Accordingly, the present invention provides methods for delivering
a recombinant adeno-associated viral (rAAV) particle containing a
rAAV vector encoding a heterologous nucleic acid to the central
nervous system of a mammal by administering the rAAV particle to
the striatum where the heterologous nucleic acid is expressed in at
least the cerebral cortex and striatum of the mammal.
[0064] The invention also provides methods for treating a CNS
disorder (e.g., Huntington's disease) in a mammal by administering
to the striatum a rAAV particle encoding a heterologous nucleic
acid that is expressed in at least the cerebral cortex and
striatum, as well as systems and kits for expression of a
heterologous nucleic acid in the cerebral cortex and striatum of a
mammal using a rAAV particle described herein. The methods in the
invention may also utilize a delivery device (e.g., a CED device)
for delivery of the rAAV particle to the striatum of a mammal, and
likewise, the systems and kits of the invention may further include
a device for delivery of the rAAV particle to the striatum of a
mammal.
I. General Techniques
[0065] The techniques and procedures described or referenced herein
are generally well understood and commonly employed using
conventional methodology by those skilled in the art, such as, for
example, the widely utilized methodologies described in Molecular
Cloning: A Laboratory Manual (Sambrook et al., 4.sup.th ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012);
Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.,
2003); the series Methods in Enzymology (Academic Press, Inc.); PCR
2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R.
Taylor eds., 1995); Antibodies, A Laboratory Manual (Harlow and
Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic
Technique and Specialized Applications (R. I. Freshney, 6.sup.th
ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (M. J.
Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell
Biology: A Laboratory Notebook (J. E. Cellis, ed., Academic Press,
1998); Introduction to Cell and Tissue Culture (J. P. Mather and P.
E. Roberts, Plenum Press, 1998); Cell and Tissue Culture:
Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,
eds., J. Wiley and Sons, 1993-8); Handbook of Experimental
Immunology (D. M. Weir and C. C. Blackwell, eds., 1996); Gene
Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,
eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al.,
eds., 1994); Current Protocols in Immunology (J. E. Coligan et al.,
eds., 1991); Short Protocols in Molecular Biology (Ausubel et al.,
eds., J. Wiley and Sons, 2002); Immunobiology (C. A. Janeway et
al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical
Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal
Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds.,
Oxford University Press, 2000); Using Antibodies: A Laboratory
Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press,
1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood
Academic Publishers, 1995); and Cancer: Principles and Practice of
Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company,
2011).
II. Definitions
[0066] A "vector," as used herein, refers to a recombinant plasmid
or virus that comprises a nucleic acid to be delivered into a host
cell, either in vitro or in vivo.
[0067] The term "polynucleotide" or "nucleic acid" as used herein
refers to a polymeric form of nucleotides of any length, either
ribonucleotides or deoxyribonucleotides. Thus, this term includes,
but is not limited to, single-, double- or multi-stranded DNA or
RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising
purine and pyrimidine bases, or other natural, chemically or
biochemically modified, non-natural, or derivatized nucleotide
bases. The backbone of the polynucleotide can comprise sugars and
phosphate groups (as may typically be found in RNA or DNA), or
modified or substituted sugar or phosphate groups. Alternatively,
the backbone of the polynucleotide can comprise a polymer of
synthetic subunits such as phosphoramidates and thus can be an
oligodeoxynucleoside phosphoramidate (P--NH.sub.2) or a mixed
phosphoramidate-phosphodiester oligomer. In addition, a
double-stranded polynucleotide can be obtained from the single
stranded polynucleotide product of chemical synthesis either by
synthesizing the complementary strand and annealing the strands
under appropriate conditions, or by synthesizing the complementary
strand de novo using a DNA polymerase with an appropriate
primer.
[0068] The terms "polypeptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues, and
are not limited to a minimum length. Such polymers of amino acid
residues may contain natural or non-natural amino acid residues,
and include, but are not limited to, peptides, oligopeptides,
dimers, trimers, and multimers of amino acid residues. Both
full-length proteins and fragments thereof are encompassed by the
definition. The terms also include post-expression modifications of
the polypeptide, for example, glycosylation, sialylation,
acetylation, phosphorylation, and the like. Furthermore, for
purposes of the present invention, a "polypeptide" refers to a
protein which includes modifications, such as deletions, additions,
and substitutions (generally conservative in nature), to the native
sequence, as long as the protein maintains the desired activity.
These modifications may be deliberate, as through site-directed
mutagenesis, or may be accidental, such as through mutations of
hosts which produce the proteins or errors due to PCR
amplification.
[0069] A "recombinant viral vector" refers to a recombinant
polynucleotide vector comprising one or more heterologous sequences
(i.e., nucleic acid sequence not of viral origin). In the case of
recombinant AAV vectors, the recombinant nucleic acid is flanked by
at least one inverted terminal repeat sequences (ITRs). In some
embodiments, the recombinant nucleic acid is flanked by two
ITRs.
[0070] A "recombinant AAV vector (rAAV vector)" refers to a
polynucleotide vector comprising one or more heterologous sequences
(i.e., nucleic acid sequence not of AAV origin) that are flanked by
at least one or two AAV inverted terminal repeat sequences (ITRs).
Such rAAV vectors can be replicated and packaged into infectious
viral particles when present in a host cell that has been infected
with a suitable helper virus (or that is expressing suitable helper
functions) and that is expressing AAV rep and cap gene products
(i.e. AAV Rep and Cap proteins). When a rAAV vector is incorporated
into a larger polynucleotide (e.g., in a chromosome or in another
vector such as a plasmid used for cloning or transfection), then
the rAAV vector may be referred to as a "pro-vector" which can be
"rescued" by replication and encapsidation in the presence of AAV
packaging functions and suitable helper functions. An rAAV vector
can be in any of a number of forms, including, but not limited to,
plasmids, linear artificial chromosomes, complexed with lipids,
encapsulated within liposomes, and encapsidated in a viral
particle; for example, an AAV particle. A rAAV vector can be
packaged into an AAV virus capsid to generate a "recombinant
adeno-associated viral particle (rAAV particle)".
[0071] "Heterologous" means derived from a genotypically distinct
entity from that of the rest of the entity to which it is compared
or into which it is introduced or incorporated. For example, a
polynucleotide introduced by genetic engineering techniques into a
different cell type is a heterologous polynucleotide (and, when
expressed, can encode a heterologous polypeptide). Similarly, a
cellular sequence (e.g., a gene or portion thereof) that is
incorporated into a viral vector is a heterologous nucleotide
sequence with respect to the vector. A heterologous nucleic acid
may refer to a nucleic acid derived from a genotypically distinct
entity from that of the rest of the entity to which it is compared
or into which it is introduced or incorporated.
[0072] The term "heterologous nucleic acid" refers to a
polynucleotide that is introduced into a cell and is capable of
being transcribed into RNA and optionally, translated and/or
expressed under appropriate conditions. In some aspects, it confers
a desired property to a cell into which it was introduced, or
otherwise leads to a desired therapeutic or diagnostic outcome. In
another aspect, it may be transcribed into a molecule that mediates
RNA interference, such as miRNA, siRNA, or shRNA.
[0073] "Chicken .beta.-actin (CBA) promoter" refers to a
polynucleotide sequence derived from a chicken .beta.-actin gene
(e.g., Gallus gallus beta actin, represented by GenBank Entrez Gene
ID 396526). As used herein, "chicken .beta.-actin promoter" may
refer to a promoter containing a cytomegalovirus (CMV) early
enhancer element, the promoter and first exon and intron of the
chicken .beta.-actin gene, and the splice acceptor of the rabbit
beta-globin gene, such as the sequences described in Miyazaki, J.
et al. (1989) Gene 79(2):269-77. As used herein, the term "CAG
promoter" may be used interchangeably. As used herein, the term
"CMV early enhancer/chicken beta actin (CAG) promoter" may be used
interchangeably.
[0074] The terms "genome particles (gp)," "genome equivalents," or
"genome copies" as used in reference to a viral titer, refer to the
number of virions containing the recombinant AAV DNA genome,
regardless of infectivity or functionality. The number of genome
particles in a particular vector preparation can be measured by
procedures such as described in the Examples herein, or for
example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039;
Veldwijk et al. (2002) Mol. Ther., 6:272-278.
[0075] The term "vector genome (vg)" as used herein may refer to
one or more polynucleotides comprising a set of the polynucleotide
sequences of a vector, e.g., a viral vector. A vector genome may be
encapsidated in a viral particle. Depending on the particular viral
vector, a vector genome may comprise single-stranded DNA,
double-stranded DNA, or single-stranded RNA, or double-stranded
RNA. A vector genome may include endogenous sequences associated
with a particular viral vector and/or any heterologous sequences
inserted into a particular viral vector through recombinant
techniques. For example, a recombinant AAV vector genome may
include at least one ITR sequence flanking a promoter, a sequence
of interest (e.g., a heterologous nucleic acid), and a
polyadenylation sequence. A complete vector genome may include a
complete set of the polynucleotide sequences of a vector. In some
embodiments, the nucleic acid titer of a viral vector may be
measured in terms of vg/mL. Methods suitable for measuring this
titer are known in the art (e.g., quantitative PCR).
[0076] The terms "infection unit (iu)," "infectious particle," or
"replication unit," as used in reference to a viral titer, refer to
the number of infectious and replication-competent recombinant AAV
vector particles as measured by the infectious center assay, also
known as replication center assay, as described, for example, in
McLaughlin et al. (1988) J. Virol., 62:1963-1973.
[0077] The term "transducing unit (tu)" as used in reference to a
viral titer, refers to the number of infectious recombinant AAV
vector particles that result in the production of a functional
heterologous nucleic acid product as measured in functional assays
such as described in Examples herein, or for example, in Xiao et
al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al. (1996)
J. Virol., 70:520-532 (LFU assay).
[0078] An "inverted terminal repeat" or "ITR" sequence is a term
well understood in the art and refers to relatively short sequences
found at the termini of viral genomes which are in opposite
orientation.
[0079] An "AAV inverted terminal repeat (ITR)" sequence, a term
well-understood in the art, is an approximately 145-nucleotide
sequence that is present at both termini of the native
single-stranded AAV genome. The outermost 125 nucleotides of the
ITR can be present in either of two alternative orientations,
leading to heterogeneity between different AAV genomes and between
the two ends of a single AAV genome. The outermost 125 nucleotides
also contains several shorter regions of self-complementarity
(designated A, A', B, B', C, C' and D regions), allowing
intrastrand base-pairing to occur within this portion of the
ITR.
[0080] A "terminal resolution sequence" or "trs" is a sequence in
the D region of the AAV ITR that is cleaved by AAV rep proteins
during viral DNA replication. A mutant terminal resolution sequence
is refractory to cleavage by AAV rep proteins.
[0081] "AAV helper functions" refer to functions that allow AAV to
be replicated and packaged by a host cell. AAV helper functions can
be provided in any of a number of forms, including, but not limited
to, helper virus or helper virus genes which aid in AAV replication
and packaging. Other AAV helper functions are known in the art such
as genotoxic agents.
[0082] A "helper virus" for AAV refers to a virus that allows AAV
(which is a defective parvovirus) to be replicated and packaged by
a host cell. A helper virus provides "helper functions" which allow
for the replication of AAV. A number of such helper viruses have
been identified, including adenoviruses, herpesviruses and,
poxviruses such as vaccinia and baculovirus. The adenoviruses
encompass a number of different subgroups, although Adenovirus type
5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses
of human, non-human mammalian and avian origin are known and are
available from depositories such as the ATCC. Viruses of the herpes
family, which are also available from depositories such as ATCC,
include, for example, herpes simplex viruses (HSV), Epstein-Barr
viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses
(PRV). Examples of adenovirus helper functions for the replication
of AAV include E1A functions, E1B functions, E2A functions, VA
functions and E4orf6 functions. Baculoviruses available from
depositories include Autographa californica nuclear polyhedrosis
virus.
[0083] A preparation of rAAV is said to be "substantially free" of
helper virus if the ratio of infectious AAV particles to infectious
helper virus particles is at least about 10.sup.2:l; at least about
10.sup.4:l, at least about 10.sup.6:l; or at least about 10.sup.8:l
or more. In some embodiments, preparations are also free of
equivalent amounts of helper virus proteins (i.e., proteins as
would be present as a result of such a level of helper virus if the
helper virus particle impurities noted above were present in
disrupted form). Viral and/or cellular protein contamination can
generally be observed as the presence of Coomassie staining bands
on SDS gels (e.g., the appearance of bands other than those
corresponding to the AAV capsid proteins VP1, VP2 and VP3).
[0084] "AAV helper functions" refer to functions that allow AAV to
be replicated and packaged by a host cell. AAV helper functions can
be provided in any of a number of forms, including, but not limited
to, helper virus or helper virus genes which aid in AAV replication
and packaging. Other AAV helper functions are known in the art such
as genotoxic agents. A "helper virus" for AAV refers to a virus
that allows AAV (which is a defective parvovirus) to be replicated
and packaged by a host cell. A number of such helper viruses have
been identified, including adenoviruses, herpesviruses and
poxviruses such as vaccinia. The adenoviruses encompass a number of
different subgroups, although Adenovirus type 5 of subgroup C (Ad5)
is most commonly used. Numerous adenoviruses of human, non-human
mammalian and avian origin are known and are available from
depositories such as the ATCC. Viruses of the herpes family, which
are also available from depositories such as ATCC, include, for
example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV),
cytomegaloviruses (CMV) and pseudorabies viruses (PRV).
[0085] "Percent (%) sequence identity" with respect to a reference
polypeptide or nucleic acid sequence is defined as the percentage
of amino acid residues or nucleotides in a candidate sequence that
are identical with the amino acid residues or nucleotides in the
reference polypeptide or nucleic acid sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity, and not considering any
conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid or nucleic
acid sequence identity can be achieved in various ways that are
within the skill in the art, for instance, using publicly available
computer software programs, for example, those described in Current
Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supp.
30, section 7.7.18, Table 7.7.1, and including BLAST, BLAST-2,
ALIGN or Megalign (DNASTAR) software. An example of an alignment
program is ALIGN Plus (Scientific and Educational Software,
Pennsylvania). Those skilled in the art can determine appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, the % amino acid sequence
identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B (which can alternatively be phrased as
a given amino acid sequence A that has or comprises a certain %
amino acid sequence identity to, with, or against a given amino
acid sequence B) is calculated as follows: 100 times the fraction
X/Y, where X is the number of amino acid residues scored as
identical matches by the sequence alignment program in that
program's alignment of A and B, and where Y is the total number of
amino acid residues in B. It will be appreciated that where the
length of amino acid sequence A is not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will
not equal the % amino acid sequence identity of B to A. For
purposes herein, the % nucleic acid sequence identity of a given
nucleic acid sequence C to, with, or against a given nucleic acid
sequence D (which can alternatively be phrased as a given nucleic
acid sequence C that has or comprises a certain % nucleic acid
sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows: 100 times the fraction W/Z,
where W is the number of nucleotides scored as identical matches by
the sequence alignment program in that program's alignment of C and
D, and where Z is the total number of nucleotides in D. It will be
appreciated that where the length of nucleic acid sequence C is not
equal to the length of nucleic acid sequence D, the % nucleic acid
sequence identity of C to D will not equal the % nucleic acid
sequence identity of D to C.
[0086] An "isolated" molecule (e.g., nucleic acid or protein) or
cell means it has been identified and separated and/or recovered
from a component of its natural environment.
[0087] An "effective amount" is an amount sufficient to effect
beneficial or desired results, including clinical results (e.g.,
amelioration of symptoms, achievement of clinical endpoints, and
the like). An effective amount can be administered in one or more
administrations. In terms of a disease state, an effective amount
is an amount sufficient to ameliorate, stabilize, or delay
development of a disease.
[0088] As used herein, the term "convection enhanced delivery
(CED)" may refer to delivery of a therapeutic agent to the CNS by
infusion at a rate in which hydrostatic pressure leads to
convective distribution. In some embodiments, the infusion is done
at a rate greater than 0.5 .mu.L/min. However, any suitable flow
rate can be used such that the intracranial pressure is maintained
at suitable levels so as not to injure the brain tissue. CED may be
accomplished, for example, by using a suitable catheter or cannula
(e.g., a step-design reflux-free cannula) through positioning the
tip of the cannula at least in close proximity to the target CNS
tissue (for example, the tip is inserted into the CNS tissue).
After the cannula is positioned, it is connected to a pump which
delivers the therapeutic agent through the cannula tip to the
target CNS tissue. A pressure gradient from the tip of the cannula
may be maintained during infusion. In some embodiments, infusion
may be monitored by a tracing agent detectable by an imaging method
such as intraoperative MRI (iMRI) or another real-time MRI
technique and/or delivered by standard stereotaxic injection
equipment and techniques (e.g., the ClearPoint.RTM. system from MRI
Interventions, Memphis, Tenn.).
[0089] As used herein, the term "poloxamer" may refer to a block
copolymer made of a chain of polyoxypropylene flanked by two chains
of polyoxyethylene. Trade names under which poloxamers may be sold
include without limitation PLURONIC.RTM. (BASF), KOLLIPHOR.RTM.
(BASF), LUTROL.RTM. (BASF), and SYNPERONIC.RTM. (Croda
International).
[0090] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0091] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (e.g., not worsening) state of disease,
preventing spread (e.g., metastasis) of disease, delay or slowing
of disease progression, amelioration or palliation of the disease
state, and remission (whether partial or total), whether detectable
or undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment.
[0092] As used herein, the term "prophylactic treatment" refers to
treatment, wherein an individual is known or suspected to have or
be at risk for having a disorder but has displayed no symptoms or
minimal symptoms of the disorder. An individual undergoing
prophylactic treatment may be treated prior to onset of
symptoms.
[0093] "Huntington's disease (HD)" refers to the progressive brain
disorder typically caused by mutations in the HTT gene (aka
huntingtin, HD or IT15). It may be characterized by symptoms
including abnormal movements (termed chorea), gradual loss of motor
function, emotional or psychiatric illnesses, and progressively
impaired cognition. Although most symptoms appear in the 30s and
40s, juvenile forms of the disease have also been observed. For
further description of HD, see OMIM Entry No. 143100, which is
hereby incorporated by reference in its entirety.
[0094] "Huntingtin (HTT)" may refer either to the gene or to a
polypeptide product thereof associated with most cases of
Huntington's disease. The normal function of huntingtin is not
fully understood. However, mutations in the huntingtin gene are
known to cause HD. These mutations are typically inherited in an
autosomal dominant fashion and involve expansion of trinucleotide
CAG repeats in the HTT gene, leading to a polyglutamine (polyQ)
tract in the Htt protein.
[0095] As used herein, a "therapeutic" agent (e.g., a therapeutic
polypeptide, nucleic acid, transgene, or the like) is one that
provides a beneficial or desired clinical result, such as the
exemplary clinical results described above. As such, a therapeutic
agent may be used in a treatment as described above.
[0096] Reference to "about" a value or parameter herein includes
(and describes) embodiments that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X."
[0097] As used herein, the singular form of the articles "a," "an,"
and "the" includes plural references unless indicated
otherwise.
[0098] It is understood that aspects and embodiments of the
invention described herein include "comprising," "consisting,"
and/or "consisting essentially of" aspects and embodiments.
III. Methods for Delivering rAAV Particles
[0099] In some aspects, the invention provides methods for
delivering a recombinant adeno-associated viral (rAAV) particle to
the central nervous system of a mammal comprising administering the
rAAV particle to the striatum, wherein the rAAV particle comprises
a rAAV vector encoding a heterologous nucleic acid that is
expressed in at least the cerebral cortex and striatum of the
mammal. In further aspects, the invention provides methods for
delivering a rAAV particle to the central nervous system of a
mammal comprising administering the rAAV particle to the striatum,
wherein the rAAV particle comprises an rAAV vector encoding a
heterologous nucleic acid that is expressed in at least the
cerebral cortex and striatum of the mammal and wherein the rAAV
particle comprises an AAV serotype 1 (AAV1) capsid. In yet further
aspects, the invention provides methods for delivering a rAAV
particle to the central nervous system of a mammal comprising
administering the rAAV particle to the striatum, wherein the rAAV
particle comprises an rAAV vector encoding a heterologous nucleic
acid that is expressed in at least the cerebral cortex and striatum
of the mammal and wherein the rAAV particle comprises an AAV
serotype 2 (AAV2) capsid. In still further aspects, the invention
provides methods for treating Huntington's disease in a mammal
comprising administering a rAAV particle to the striatum, wherein
the rAAV particle comprises a rAAV vector encoding a heterologous
nucleic acid that is expressed in at least the cerebral cortex and
striatum of the mammal. In some embodiments, the mammal is a
human.
[0100] Certain aspects of the present disclosure relate to
administration of a rAAV particle to one or more regions of the
central nervous system (CNS). In some embodiments, the rAAV
particle is administered to the striatum. The striatum is known as
a region of the brain that receives inputs from the cerebral cortex
(the term "cortex" may be used interchangeably herein) and sends
outputs to the basal ganglia (the striatum is also referred to as
the striate nucleus and the neostriatum). As described above, the
striatum controls both motor movements and emotional
control/motivation and has been implicated in many neurological
diseases, such as Huntington's disease. Several cell types of
interest are located in the striatum, including without limitation
spiny projection neurons (also known as medium spiny neurons),
GABAergic interneurons, and cholinergic interneurons. Medium spiny
neurons make up most of the striatal neurons. These neurons are
GABAergic and express dopamine receptors. Each hemisphere of the
brain contains a striatum.
[0101] Important substructures of the striatum include the caudate
nucleus and the putamen. In some embodiments, the rAAV particle is
administered to the caudate nucleus (the term "caudate" may be used
interchangeably herein). The caudate nucleus is known as a
structure of the dorsal striatum. The caudate nucleus has been
implicated in control of functions such as directed movements,
spatial working memory, memory, goal-directed actions, emotion,
sleep, language, and learning. Each hemisphere of the brain
contains a caudate nucleus.
[0102] In some embodiments, the rAAV particle is administered to
the putamen. Along with the caudate nucleus, the putamen is known
as a structure of the dorsal striatum. The putamen comprises part
of the lenticular nucleus and connects the cerebral cortex with the
substantia nigra and the globus pallidus. Highly integrated with
many other structures of the brain, the putamen has been implicated
in control of functions such as learning, motor learning, motor
performance, motor tasks, and limb movements. Each hemisphere of
the brain contains a putamen.
[0103] rAAV particles may be administered to one or more sites of
the striatum. In some embodiments, the rAAV particle is
administered to the putamen and the caudate nucleus of the
striatum. In some embodiments, the rAAV particle is administered to
the putamen and the caudate nucleus of each hemisphere of the
striatum. In some embodiments, the rAAV particle is administered to
at least one site in the caudate nucleus and two sites in the
putamen.
[0104] In some embodiments, the rAAV particle is administered to
one hemisphere of the brain. In some embodiments, the rAAV particle
is administered to both hemispheres of the brain. For example, in
some embodiments, the rAAV particle is administered to the putamen
and the caudate nucleus of each hemisphere of the striatum. In some
embodiments, the composition containing rAAV particles is
administered to the striatum of each hemisphere. In other
embodiments, the composition containing rAAV particles is
administered to striatum of the left hemisphere or the striatum of
the right hemisphere and/or the putamen of the left hemisphere or
the putamen of the right hemisphere. In some embodiments, the
composition containing rAAV particles is administered to any
combination of the caudate nucleus of the left hemisphere, the
caudate nucleus of the right hemisphere, the putamen of the left
hemisphere and the putamen of the right hemisphere.
[0105] In some embodiments, the composition containing rAAV
particles is administered to more than one location simultaneously
or sequentially. In some embodiments, multiple injections of the
composition containing rAAV particles are no more than about any of
one hour, two hours, three hours, four hours, five hours, six
hours, nine hours, twelve hours or 24 hours apart. In some
embodiments, multiple injections of the composition containing rAAV
particles are more than about 24 hours apart.
[0106] Generally, from about 14 to about 1 mL of a composition of
the invention can be delivered (e.g., from about 100 .mu.L to about
500 .mu.L of a composition). In some embodiments, the amount of the
composition delivered to the putamen is greater than the volume
delivered to the caudate nucleus. In some embodiments, the amount
of the composition delivered to the putamen is about twice the
volume delivered to the caudate nucleus. In other embodiments, the
amount of the composition delivered to the putamen is about any of
1.times., 1.25.times., 1.5.times.. 1.75.times., 2.times.,
2.25.times., 2.5.times.. 2.75.times., 3.times., 3.5.times.,
4.times., 4.5.times., 5.times. or 10.times. (or any ratio
therebetween) the volume delivered to the caudate nucleus. For
example, in some embodiments, the ratio of rAAV particles
administered to the putamen to rAAV particles administered to the
caudate nucleus is at least about 2:1 (e.g., about 30 .mu.L of the
composition is administered to the caudate nucleus of each
hemisphere and about 60 .mu.L of the composition is administered to
the putamen of each hemisphere). In some embodiments, about 20
.mu.L to about 50 .mu.L of the composition (or any amount
therebetween) is administered to the caudate nucleus of each
hemisphere, and about 40 .mu.L to about 100 .mu.L of the
composition (or any amount therebetween) is administered to the
putamen of each hemisphere. In some embodiments, the volume of the
composition administered to the caudate nucleus of each hemisphere
is less than about any of the following volumes (in .mu.L): 50, 45,
40, 35, 30, or 25. In some embodiments, the volume of the
composition administered to the caudate nucleus of each hemisphere
is greater than about any of the following volumes (in .mu.L): 20,
25, 30, 35, 40, or 45. That is, the volume of the composition
administered to the caudate nucleus of each hemisphere can be any
of a range of volumes having an upper limit of 50, 45, 40, 35, 30,
or 25 and an independently selected lower limit of 20, 25, 30, 35,
40, or 45, wherein the lower limit is less than the upper limit. In
some embodiments, the volume of the composition administered to the
putamen of each hemisphere is less than about any of the following
volumes (in .mu.L): 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, or
45. In some embodiments, the volume of the composition administered
to the putamen of each hemisphere is greater than about any of the
following volumes (in .mu.L): 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, or 95. That is, the volume of the composition administered
to the putamen of each hemisphere can be any of a range of volumes
having an upper limit of 100, 95, 90, 85, 80, 75, 70, 65, 60, 55,
50, or 45 and an independently selected lower limit of 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, or 95, wherein the lower limit is
less than the upper limit.
[0107] In some embodiments, the composition is administered to the
striatum at a rate of greater than 1 .mu.L/min to about 5
.mu.L/min. In some embodiments, the composition is administered to
the caudate nucleus and the putamen at a rate of greater than 1
.mu.L/min to about 5 .mu.L/min. In some embodiments, the
composition is administered to the striatum (the caudate nucleus
and/or the putamen) at a rate of greater than about any of 1
.mu.L/min, 2 .mu.L/min, 3 .mu.L/min, 4 .mu.L/min, 5 .mu.L/min, 6
.mu.L/min, 7 .mu.L/min, 8 .mu.L/min, 9 .mu.L/min, or 10 .mu.L/min.
In some embodiments, the composition is administered to the
striatum (the caudate nucleus and/or the putamen) at a rate of any
of about 1 .mu.L/min to about 10 .mu.L/min, about 1 .mu.L/min to
about 9 .mu.L/min, about 1 .mu.L/min to about 8 .mu.L/min, about 1
.mu.L/min to about 7 .mu.L/min, about 1 .mu.L/min to about 6
.mu.L/min, about 1 .mu.L/min to about 5 .mu.L/min, about 1
.mu.L/min to about 4 .mu.L/min, about 1 .mu.L/min to about 3
.mu.L/min, about 1 .mu.L/min to about 2 .mu.L/min, about 2
.mu.L/min to about 10 .mu.L/min, about 2 .mu.L/min to about 9
.mu.L/min, about 2 .mu.L/min to about 8 .mu.L/min, about 2
.mu.L/min to about 7 .mu.L/min, about 2 .mu.L/min to about 6
.mu.L/min, about 2 .mu.L/min to about 5 .mu.L/min, about 2
.mu.L/min to about 4 .mu.L/min, about 2 .mu.L/min to about 3
.mu.L/min, about 3 .mu.L/min to about 10 .mu.L/min, about 3
.mu.L/min to about 9 .mu.L/min, about 3 .mu.L/min to about 8
.mu.L/min, about 3 .mu.L/min to about 7 .mu.L/min, about 3
.mu.L/min to about 6 .mu.L/min, about 3 .mu.L/min to about 5
.mu.L/min, about 3 .mu.L/min to about 4 .mu.L/min, about 4
.mu.L/min to about 10 .mu.L/min, about 4 .mu.L/min to about 9
.mu.L/min, about 4 .mu.L/min to about 8 .mu.L/min, about 4
.mu.L/min to about 7 .mu.L/min, about 4 .mu.L/min to about 6
.mu.L/min, about 4 .mu.L/min to about 5 .mu.L/min, about 5
.mu.L/min to about 10 .mu.L/min, about 5 .mu.L/min to about 9
.mu.L/min, about 5 .mu.L/min to about 8 .mu.L/min, about 5
.mu.L/min to about 7 .mu.L/min, about 5 .mu.L/min to about 6
.mu.L/min, about 6 .mu.L/min to about 10 .mu.L/min, about 6
.mu.L/min to about 9 .mu.L/min, about 6 .mu.L/min to about 8
.mu.L/min, about 6 .mu.L/min to about 7 .mu.L/min, about 7
.mu.L/min to about 10 .mu.L/min, about 7 .mu.L/min to about 9
.mu.L/min, about 7 .mu.L/min to about 8 .mu.L/min, about 8
.mu.L/min to about 10 .mu.L/min, about 8 .mu.L/min to about 9
.mu.L/min, or about 9 .mu.L/min to about 10 .mu.L/min. in some
embodiments, the composition is administered in incremental
increases in flow rate during delivery (i.e., "stepping").
[0108] In some embodiments, administration of the rAAV particle is
performed once. In other embodiments, administration of the rAAV
particle is performed more than once. One of skill in the art may
determine how many times to perform administration of the rAAV
particle based in part on, e.g., the disorder being treated and/or
the patient response to treatment.
[0109] In some embodiments, the methods comprise administration to
CNS an effective amount of recombinant viral particles to the
striatum, wherein the rAAV particle comprises a rAAV vector
encoding a heterologous nucleic acid that is expressed in at least
the cerebral cortex and striatum. In some embodiments, the viral
titer of the rAAV particles is at least about any of
5.times.10.sup.12, 6.times.10.sup.12, 7.times.10.sup.12,
8.times.10.sup.12, 9.times.10.sup.12, 10.times.10.sup.12,
11.times.10.sup.12, 15.times.10.sup.12, 20.times.10.sup.12,
25.times.10.sup.12, 30.times.10.sup.12, or 50.times.10.sup.12
genome copies/mL. In some embodiments, the viral titer of the rAAV
particles is about any of 5.times.10.sup.12 to 6.times.10.sup.12,
6.times.10.sup.12 to 7.times.10.sup.12, 7.times.10.sup.12 to
8.times.10.sup.12, 8.times.10.sup.12 to 9.times.10.sup.12,
9.times.10.sup.12 to 10.times.10.sup.12, 10.times.10.sup.12 to
11.times.10.sup.12, 11.times.10.sup.12 to 15.times.10.sup.12,
15.times.10.sup.12 to 20.times.10.sup.12, 20.times.10.sup.12 to
25.times.10.sup.12, 25.times.10.sup.12 to 30.times.10.sup.12,
30.times.10.sup.12 to 50.times.10.sup.12, or 50.times.10.sup.12 to
100.times.10.sup.12 genome copies/mL. In some embodiments, the
viral titer of the rAAV particles is about any of 5.times.10.sup.12
to 10.times.10.sup.12, 10.times.10.sup.12 to 25.times.10.sup.12, or
25.times.10.sup.12 to 50.times.10.sup.12genome copies/mL. In some
embodiments, the viral titer of the rAAV particles is at least
about any of 5.times.10.sup.9, 6.times.10.sup.9, 7.times.10.sup.9,
8.times.10.sup.9, 9.times.10.sup.9, 10.times.10.sup.9,
11.times.10.sup.9, 15.times.10.sup.9, 20.times.10.sup.9,
25.times.10.sup.9, 30.times.10.sup.9, or 50.times.10.sup.9
transducing units/mL. In some embodiments, the viral titer of the
rAAV particles is about any of 5.times.10.sup.9 to
6.times.10.sup.9, 6.times.10.sup.9 to 7.times.10.sup.9,
7.times.10.sup.9 to 8.times.10.sup.9, 8.times.10.sup.9 to
9.times.10.sup.9, 9.times.10.sup.9 to 10.times.10.sup.9,
10.times.10.sup.9 to 11.times.10.sup.9, 11.times.10.sup.9 to
15.times.10.sup.9, 15.times.10.sup.9 to 20.times.10.sup.9,
20.times.10.sup.9 to 25.times.10.sup.9, 25.times.10.sup.9 to
30.times.10.sup.9, 30.times.10.sup.9 to 50.times.10.sup.9 or
50.times.10.sup.9 to 100.times.10.sup.9 transducing units/mL. In
some embodiments, the viral titer of the rAAV particles is about
any of 5.times.10.sup.9 to 10.times.10.sup.9, 10.times.10.sup.9 to
15.times.10.sup.9, 15.times.10.sup.9 to 25.times.10.sup.9, or
25.times.10.sup.9 to 50.times.10.sup.9 transducing units/mL. In
some embodiments, the viral titer of the rAAV particles is at least
any of about 5.times.10.sup.10, 6.times.10.sup.10,
7.times.10.sup.10, 8.times.10.sup.10, 9.times.10.sup.10,
10.times.10.sup.10, 11.times.10.sup.10, 15.times.10.sup.10,
20.times.10.sup.10, 25.times.10.sup.10, 30.times.10.sup.10,
40.times.10.sup.10, or 50.times.10.sup.10 infectious units/mL. In
some embodiments, the viral titer of the rAAV particles is at least
any of about 5.times.10.sup.10 to 6.times.10.sup.10,
6.times.10.sup.10 to 7.times.10.sup.10, 7.times.10.sup.10 to
8.times.10.sup.10, 8.times.10.sup.10 to 9.times.10.sup.10,
9.times.10.sup.10 to 10.times.10.sup.10, 10.times.10.sup.10 to
11.times.10.sup.10, 11.times.10.sup.10 to 15.times.10.sup.10,
15.times.10.sup.10 to 20.times.10.sup.10, 20.times.10.sup.10 to
25.times.10.sup.10, 25.times.10.sup.10 to 30.times.10.sup.10,
30.times.10.sup.10 to 40.times.10.sup.10, 40.times.10.sup.10 to
50.times.10.sup.10, or 50.times.10.sup.10 to 100.times.10.sup.10
infectious units/mL. In some embodiments, the viral titer of the
rAAV particles is at least any of about 5.times.10.sup.10 to
10.times.10.sup.10, 10.times.10.sup.10 to 15.times.10.sup.10,
15.times.10.sup.10 to 25.times.10.sup.10, or 25.times.10.sup.10 to
50.times.10.sup.10 infectious units/mL.
[0110] In some embodiments, the methods comprise administration to
CNS an effective amount of recombinant viral particles to the
striatum, wherein the rAAV particle comprises a rAAV vector
encoding a heterologous nucleic acid that is expressed in at least
the cerebral cortex and striatum. In some embodiments, the dose of
viral particles administered to the individual is at least about
any of 1.times.10.sup.8 to about 1.times.10.sup.13 genome copies/kg
of body weight. In some embodiments, the dose of viral particles
administered to the individual is about 1.times.10.sup.8 to
1.times.10.sup.13 genome copies/kg of body weight.
[0111] In some embodiments, the methods comprise administration to
CNS an effective amount of recombinant viral particles to the
striatum, wherein the rAAV particle comprises a rAAV vector
encoding a heterologous nucleic acid that is expressed in at least
the cerebral cortex and striatum. In some embodiments, the total
amount of viral particles administered to the individual is at
least about 1.times.10.sup.9 to about 1.times.10.sup.14 genome
copies. In some embodiments, the total amount of viral particles
administered to the individual is about 1.times.10.sup.9 to about
1.times.10.sup.14 genome copies.
[0112] Compositions of the invention (e.g., rAAV particles) can be
used either alone or in combination with one or more additional
therapeutic agents for treating any or all of the disorders
described herein. The interval between sequential administration
can be in terms of at least (or, alternatively, less than) minutes,
hours, or days.
IV. Expression Constructs
[0113] In some aspects, the invention provides methods for
delivering rAAV particles to the CNS of a mammal by administering
the rAAV particles to the striatum. In some embodiments, the rAAV
particles comprise a rAAV vector. The rAAV vector may encode a
heterologous nucleic acid, (e.g., a heterologous nucleic acid
expressed in at least the cerebral cortex and striatum). rAAV
vectors are described in greater detail infra.
[0114] In some embodiments, the rAAV vector encodes a heterologous
nucleic acid. In some embodiments, a heterologous nucleic acid may
encode a therapeutic polypeptide or therapeutic nucleic acid. A
therapeutic polypeptide or therapeutic nucleic acid may be used,
for example, to ameliorate a symptom, prevent or delay progression,
and/or provide a treatment of a disorder (e.g., a disorder
described herein). In some embodiments, the therapeutic polypeptide
or the therapeutic nucleic acid is used to treat a disorder of the
CNS, as described in more detail below.
[0115] The heterologous nucleic acid may be expressed in one or
more regions of interest within the CNS. For example, in some
embodiments, the heterologous nucleic acid is expressed in at least
the cerebral cortex and striatum. The heterologous nucleic acid may
be capable of expression ubiquitously throughout the CNS, or it may
be expressed in a subset of CNS cells.
[0116] In some embodiments, the heterologous nucleic acid is
expressed in the frontal cortex, occipital cortex, and/or layer IV
of the mammal. The cerebral cortex is known as the outer layer of
the mammalian brain important for language, consciousness, memory,
attention, and awareness. The cerebral cortex is subdivided into a
number of different components and regions due to its extensive
anatomy and complex functions. It may be divided into left and
right hemispheres. In addition, it contains four gross lobes:
frontal, parietal, temporal, and occipital. Frontal cortex may
refer to the frontal lobe of the cortex and is known to provide a
wide range of neurological functions related to non-task-based
memory, social interactions, decision making, and other complex
cognitive functions. Occipital cortex may refer to the occipital
lobe of the cortex and is known to be involved in visual
processing. Parietal cortex may refer to the parietal lobe of the
cortex and is known to be involved in language processing,
proprioception, and sensory inputs related to touch. Temporal
cortex may refer to the temporal lobe of the cortex and is known to
be involved in language, memory, and emotional association.
[0117] In addition, three general types of areas of the cortex are
described: sensory, motor, and association. These may be divided
into 5 functional subdivisions: primary motor cortex (involved in
muscle control), premotor cortex (higher order motor areas that
command primary motor areas), association areas (e.g.,
parietal-temporal-occipital or prefrontal; these areas are involved
in planning, memory, attention, and other higher cognitive tasks
and assume the majority of the human cortex), higher order areas
(sensory processing), and primary sensory areas (e.g., auditory,
visual, and somatosensory). In some embodiments, the heterologous
nucleic acid is expressed in the prefrontal association cortical
areas, the premotor cortex, the primary somatosensory cortical
areas, sensory motor cortex, parietal cortex, occipital cortex,
and/or primary motor cortex.
[0118] In addition, the cerebral cortex may be divided into
different cortical layers (moving from superficial to deep), each
containing a characteristic pattern of neuronal connectivities and
cell types. These layers may be divided into supragranular layers
(layers internal granular (IV), and infragranular (V and VI).
Supragranular layers typically project to other cortical layers,
whereas infragranular layers receive input from supragranular
layers and send output to structures outside the cortex (e.g.,
motor, sensory, and thalamic regions). Layer V contains pyramidal
neurons with axons that connect to subcortical structures like the
basal ganglia. Layer V neurons in the primary motor cortex also
form the corticospinal tract that is critical for voluntary motor
control. Layer IV receives inputs from the thalamus and connects to
the rest of the column, thereby providing critical functions
related to integration of the thalamus and cortex. Characteristic
cells of layer IV include stellate cells (e.g., spiny stellate
cells) and pyramidal neurons.
[0119] In some embodiments, the rAAV particle undergoes retrograde
or anterograde transport in the cerebral cortex. Retrograde
transport refers to the phenomenon by which cargo (e.g., rAAV
particles) is moved from a neuronal process (e.g., an axon) to the
cell body. Anterograde transport refers to movement from the cell
body to the cell membrane (e.g., a synapse). Retrograde transport
of AAV particles is thought to occur via receptor-mediated
internalization at the axon terminal, followed by
microtubule-mediated transport to the nucleus (see, e.g., Kaspar et
al., (2002) Mol. Ther. 5:50-56; Boulis et al., (2003) Neurobiol.
Dis. 14:535-541; Kaspar et al., (2003) Science 301:839-842). It is
known that the striatum contains projections from other brain
regions, such as regions of the cortex. Both anterograde and
retrograde transport may allow rAAV particles to be distributed
throughout the brain, such as between the cortex and thalamus (see,
e.g., Kells, A. P. et al. (2009) Proc. Natl. Acad. Sci.
106:2407-2411). Therefore, without wishing to be bound to theory,
it is thought that injection of AAV particles into one brain region
(e.g., the striatum, caudate nucleus, and/or putamen) may allow the
AAV particles to be delivered to other areas of the brain (e.g.,
the cortex) through retrograde transport.
[0120] In some embodiments, the heterologous nucleic acid is
further expressed in the thalamus, substantia nigra and/or
hippocampus. As described above, mechanisms such as anterograde
and/or retrograde transport may allow rAAV particles injected into
the cerebral cortex and/or striatum to be distributed to other
regions of the brain, particularly those that connect to the
cortex. The thalamus is between the cortex and midbrain, sends
signals (e.g., sensory and motor) to the cortex from subcortical
areas, and plays a role in alertness and sleep. The thalamus also
connects to the hippocampus, part of the limbic system and a
critical mediator of long-term memory consolidation. Part of the
basal ganglia, the substantia nigra contains many dopaminergic
neurons and is important for movement and reward. CNS disorders
like Parkinson's disease are associated with loss of dopaminergic
neurons in the substantia nigra. It further provides dopamine to
the striatum that is critical for proper striatal function.
[0121] In some aspects, the invention provides rAAV vectors for use
in methods of preventing or treating one or more gene defects
(e.g., heritable gene defects, somatic gene alterations, and the
like) in a mammal, such as for example, a gene defect that results
in a polypeptide deficiency or polypeptide excess in a subject, or
for treating or reducing the severity or extent of deficiency in a
subject manifesting a CNS-associated disorder linked to a
deficiency in such polypeptides in cells and tissues. In some
embodiments, methods involve administration of a rAAV vector that
encodes one or more therapeutic peptides, polypeptides, functional
RNAs, inhibitory nucleic acids, shRNAs, microRNAs, antisense
nucleotides, etc. in a pharmaceutically-acceptable carrier to the
subject in an amount and for a period of time sufficient to treat
the CNS-associated disorder in the subject having or suspected of
having such a disorder.
[0122] A rAAV vector may comprise as a transgene, a nucleic acid
encoding a protein or functional RNA that modulates or treats a
CNS-associated disorder. The following is a non-limiting list of
genes associated with CNS-associated disorders: neuronal apoptosis
inhibitory protein (NAIP), nerve growth factor (NGF), glial-derived
growth factor (GDNF), brain-derived growth factor (BDNF), ciliary
neurotrophic factor (CNTF), tyrosine hydroxlase (TM,
GTP-cyclohydrolase (GTPCH), aspartoacylase (ASPA), superoxide
dismutase (SOD1) and amino acid decarboxylase (AADC). For example,
a useful transgene in the treatment of Parkinson's disease encodes
TH, which is a rate limiting enzyme in the synthesis of dopamine. A
transgene encoding GTPCII, which generates the TII cofactor
tetrahydrobiopterin, may also be used in the treatment of
Parkinson's disease. A transgene encoding GDNF or BDNF, or AADC,
which facilitates conversion of L-Dopa to DA, may also be used for
the treatment of Parkinson's disease. For the treatment of ALS, a
useful transgene may encode: GDNF, BDNF or CNTF. Also for the
treatment of ALS, a useful transgene may encode a functional RNA,
e.g., shRNA, miRNA, that inhibits the expression of SOD1. For the
treatment of ischemia a useful transgene may encode NAIP or NGF. A
transgene encoding Beta-glucuronidase (GUS) may be useful for the
treatment of certain lysosomal storage diseases (e.g.,
Mucopolysacharidosis type VII (MPS VII)). A transgene encoding a
prodrug activation gene, e.g., HSV-Thymidine kinase which converts
ganciclovir to a toxic nucleotide which disrupts DNA synthesis and
leads to cell death, may be useful for treating certain cancers,
e.g., when administered in combination with the prodrug. A
transgene encoding an endogenous opioid, such a .beta.-endorphin
may be useful for treating pain. Other examples of transgenes that
may be used in the rAAV vectors of the invention will be apparent
to the skilled artisan (See, e.g., Costantini L C, et al., Gene
Therapy (2000) 7, 93-109).
[0123] In some embodiments, the heterologous nucleic acid may
encode a therapeutic nucleic acid. In some embodiments, a
therapeutic nucleic acid may include without limitation an siRNA,
an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a
DNAzyme. As such, a therapeutic nucleic acid may encode an RNA that
when transcribed from the nucleic acids of the vector can treat a
disorder of the invention (e.g., a disorder of the CNS) by
interfering with translation or transcription of an abnormal or
excess protein associated with a disorder of the invention. For
example, the nucleic acids of the invention may encode for an RNA
which treats a disorder by highly specific elimination or reduction
of mRNA encoding the abnormal and/or excess proteins. Therapeutic
RNA sequences include RNAi, small inhibitory RNA (siRNA), micro RNA
(miRNA), and/or ribozymes (such as hammerhead and hairpin
ribozymes) that can treat disorders by highly specific elimination
or reduction of mRNA encoding the abnormal and/or excess
proteins.
[0124] In some embodiments, the heterologous nucleic acid may
encode a therapeutic polypeptide. A therapeutic polypeptide may,
e.g., supply a polypeptide and/or enzymatic activity that is absent
or present at a reduced level in a cell or organism. Alternatively,
a therapeutic polypeptide may supply a polypeptide and/or enzymatic
activity that indirectly counteracts an imbalance in a cell or
organism. For example, a therapeutic polypeptide for a disorder
related to buildup of a metabolite caused by a deficiency in a
metabolic enzyme or activity may supply a missing metabolic enzyme
or activity, or it may supply an alternate metabolic enzyme or
activity that leads to reduction of the metabolite. A therapeutic
polypeptide may also be used to reduce the activity of a
polypeptide (e.g., one that is overexpressed, activated by a
gain-of-function mutation, or whose activity is otherwise
misregulated) by acting, e.g., as a dominant-negative
polypeptide.
[0125] In some embodiments, the therapeutic polypeptide or
therapeutic nucleic acid is used to treat a disorder of the CNS.
Without wishing to be bound to theory, it is thought that a
therapeutic polypeptide or therapeutic nucleic acid may be used to
reduce or eliminate the expression and/or activity of a polypeptide
whose gain-of-function has been associated with a disorder, or to
enhance the expression and/or activity of a polypeptide to
complement a deficiency that has been associated with a disorder
(e.g., a mutation in a gene whose expression shows similar or
related activity). Non-limiting examples of CNS disorders of the
invention that may be treated by a therapeutic polypeptide or
therapeutic nucleic acid of the invention (exemplary genes that may
be targeted or supplied are provided in parenthesis for each
disorder) include stroke (e.g., caspase-3, Beclin1, Ask1, PAR1,
HIF1.alpha., PUMA, and/or any of the genes described in Fukuda, A.
M. and Badaut, J. (2013) Genes (Basel) 4:435-456), Huntington's
disease (mutant HTT), epilepsy (e.g., SCN1A, NMDAR, ADK, and/or any
of the genes described in Boison, D. (2010) Epilepsia
51:1659-1668), Parkinson's disease (alpha-synuclein), Lou Gehrig's
disease (also known as amyotrophic lateral sclerosis; SOD1),
Alzheimer's disease (tau, amyloid precursor protein), corticobasal
degeneration or CBD (tau), corticogasal ganglionic degeneration or
CBGD (tau), frontotemporal dementia or FTD (tau), progressive
supranuclear palsy or PSP (tau), multiple system atrophy or MSA
(alpha-synuclein), cancer of the brain (e.g., a mutant or
overexpressed oncogene implicated in brain cancer), and lysosomal
storage diseases (LSD). Disorders of the invention may include
those that involve large areas of the cortex, e.g., more than one
functional area of the cortex, more than one lobe of the cortex,
and/or the entire cortex. Other non-limiting examples of disorders
of the invention that may be treated by a therapeutic polypeptide
or therapeutic nucleic acid of the invention include traumatic
brain injury, enzymatic dysfunction disorders, psychiatric
disorders (including post-traumatic stress syndrome),
neurodegenerative diseases, and cognitive disorders (including
dementias, autism, and depression). Enzymatic dysfunction disorders
include without limitation leukodystrophies (including Canavan's
disease) and any of the lysosomal storage diseases described
below.
[0126] In some embodiments, the therapeutic polypeptide or
therapeutic nucleic acid is used to treat a lysosomal storage
disease. As is commonly known in the art, lysosomal storage disease
are rare, inherited metabolic disorders characterized by defects in
lysosomal function. Such disorders are often caused by a deficiency
in an enzyme required for proper mucopolysaccharide, glycoprotein,
and/or lipid metabolism, leading to a pathological accumulation of
lysosomally stored cellular materials. Non-limiting examples of
lysosomal storage diseases of the invention that may be treated by
a therapeutic polypeptide or therapeutic nucleic acid of the
invention (exemplary genes that may be targeted or supplied are
provided in parenthesis for each disorder) include Gaucher disease
type 2 or type 3 (acid beta-glucosidase, GBA), GM1 gangliosidosis
(beta-galactosidase-1, GLB1), Hunter disease (iduronate
2-sulfatase, IDS), Krabbe disease (galactosylceramidase, GALC), a
mannosidosis disease (a mannosidase, such as alpha-D-mannosidase,
MAN2B1), .beta. mannosidosis disease (beta-mannosidase, MANBA),
metachromatic leukodystrophy disease (pseudoarylsulfatase A, ARSA),
mucolipidosisII/III disease
(N-acetylglucosamine-1-phosphotransferase, GNP TAB), Niemann-Pick A
disease (acid sphingomyelinase, ASM), Niemann-Pick C disease
(Niemann-Pick C protein, NPC1), Pompe disease (acid
alpha-1,4-glucosidase, GAA), Sandhoff disease (hexosaminidase beta
subunit, HERB), Sanfillipo A disease (N-sulfoglucosamine
sulfohydrolase, MPS3A), Sanfillipo B disease
(N-alpha-acetylglucosaminidase, NAGLU), Sanfillipo C disease
(heparin acetyl-CoA:alpha-glucosaminidase N-acetyltransferase,
MPS3C), Sanfillipo D disease (N-acetylglucosamine-6-sulfatase,
GNS), Schindler disease (alpha-N-acetylgalactosaminidase, NAGA),
Sly disease (beta-glucuronidase, GUSB), Tay-Sachs disease
(hexosaminidase alpha subunit, HEXA), and Wolman disease (lysosomal
acid lipase, LIPA).
[0127] Additional lysosomal storage diseases, as well as the
defective enzyme associated with each disease, are listed in Table
1 below. In some embodiments, a disease listed in the table below
is treated by a therapeutic polypeptide or therapeutic nucleic acid
of the invention that complements or otherwise compensates for the
corresponding enzymatic defect.
TABLE-US-00001 TABLE 1 Lysosomal storage disorders and associated
defective enzymes. Lysosomal storage disease Defective enzyme
Aspartylglusoaminuria Aspartylglucosaminidase Fabry
Alpha-galactosidase A Infantile Batten Disease (CNL1) Palmitoyl
protein thioesterase Classic Late Infantile Batten Tripeptidyl
peptidase Disease (CNL2) Juvenile Batten Disease (CNL3) Lysosomal
transmembrane protein Batten, other forms (CNL4-CNL8) multiple gene
products Cystinosis Cysteine transporter Farber Acid ceramidase
Fucosidosis Acid alpha-L-fucosidase Galactosidosialidosis
Protective protein/cathepsin A Gaucher types 1, 2, and 3 Acid
beta-glucosidase GM1 gangliosidosis Acid beta-galactosidase Hunter
Iduronate-2-sulfatase Hurler-Scheie Alpha-L-iduronidase Krabbe
Galactocerebrosidase alpha-mannosidosis Acid alpha-mannosidase
beta-mannosidosis Acid beta-mannosidase Maroteaux-Lamy
Arylsulfatase B Metachromatic leukodystrophy Arylsulfatase A
Morquio A N-acetylgalactosamine-6-sulfate Morquio B Acid
beta-galactosidase Mucolipidosis II/III N-acetylglucosamine-1-
phosphotransferase Niemann-Pick A, B Acid sphingomyelinase
Niemann-Pick C NPC-1 Pompe acid alpha-glucosidase Sandhoff
beta-hexosaminidase B Sanfilippo A Heparan N-sulfatase Sanfilippo B
alpha-N-acetylglucosaminidase Sanfilippo C Acetyl-CoA:
alpha-glucoasaminide N-acetyltransferase Sanfilippo D
N-acetylglucosamine-6-sulfate Schindler disease
alpha-N-acetylgalactosaminidase Schindler-Kanzaki
alpha-N-acetylgalactosaminidase Sialidosis alpha-neuramidase Sly
beta-glucuronidase Tay-Sachs beta-hexosaminidase A Wolman Acid
lipase
[0128] As such, in some embodiments, the therapeutic polypeptide is
caspase-3, Beclin1, Ask1, PAR1, HIF1.alpha., PUMA, SCN1A, NMDAR,
ADK, alpha-synuclein, SOD1, acid beta-glucosidase (GBA),
beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS),
galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase
(MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA),
N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid
sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid
alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB,
N-sulfoglucosamine sulfohydrolase (MPS3A),
N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA,
alpha-glucosaminidase N-acetyltransferase (MPS3C),
N-acetylglucosamine-6-sulfatase (GNS),
alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB),
hexosaminidase alpha subunit (HEXA), huntingtin (HTT), or lysosomal
acid lipase (LIPA). The therapeutic polypeptide may increase or
decrease the function of the target polypeptide in the subject
(e.g., it may supply the missing function in a lysosomal storage
disease, or reduce the level of alpha-synuclein in MSA, such as by
blocking its function or dysfunction). In some embodiments, the
therapeutic nucleic acid is caspase-3, Beclin1, Ask1, PAR1,
HIF1.alpha., PUMA, SCN1A, NMDAR, ADK, alpha-synuclein, SOD1, acid
beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate
2-sulfatase (IDS), galactosylceramidase (GALC), a mannosidase,
alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA),
pseudoarylsulfatase A (ARSA),
N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid
sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid
alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB,
N-sulfoglucosamine sulfohydrolase (MPS3A),
N-alpha-acetylglucosaminidase (NAGLU), heparin acetyl-CoA,
alpha-glucosaminidase N-acetyltransferase (MPS3C),
N-acetylglucosamine-6-sulfatase (GNS),
alpha-N-acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB),
hexosaminidase alpha subunit (HEXA), or lysosomal acid lipase
(LIPA). The therapeutic nucleic acid may increase or decrease the
function of the target polypeptide in the subject (e.g., it may
supply the missing function in a lysosomal storage disease, or
reduce the level of alpha-synuclein in MSA, such as by RNAi).
[0129] An exemplary disease for which AAV expression in the cortex
and striatum may be useful is Huntington's disease (HD).
Huntington's disease is caused by a CAG repeat expansion mutation
that encodes an elongated polyglutamine (polyQ) repeat in the
mutant huntingtin protein (mHTT). HD is a particularly attractive
target for DNA- and RNA-based therapies as it is an autosomal
dominant disease resulting from mutation on a single allele. AAV
vectors provide an ideal delivery system for nucleic acid
therapeutics and allow for long lasting and continuous expression
of these huntingtin lowering molecules in the brain. To achieve
maximal clinical efficacy in HD, delivery to both the striatum and
cortex will likely be required. Postmortem analysis of HD patient
brains revealed extensive medium spiny neuronal loss in the
striatum, in addition to loss of pyramidal neurons in the cerebral
cortex and hippocampus. It was recently shown using conditional
transgenic mouse models of HD that genetically reducing mHTT
expression in neuronal populations in the striatum and cortex
provides significantly more efficacy than reducing mHTT in either
site alone (Wang et al., (2014) Nature medicine 20:536-541).
Together, this evidence suggests that delivery of gene therapy
agents to both striatal and cortical regions may be ideal for
maximal therapeutic efficacy.
[0130] The use of gene therapy vectors to deliver biologics to
critical brain regions implicated in Huntington's disease
pathogenesis has been a challenge, due in large part to the
physical constraints of effectively delivering a vector
specifically to the striatum and the cerebral cortex. Although
multiple direct infusions can be effective in small animal brains,
as the architecture and volume of brain tissue increases in
primates, it becomes more difficult to achieve widespread striatal
and cortical delivery through single site infusion. Therefore, the
inventors' discovery that striatal administration can achieve
widespread rAAV distribution, including the cortex and striatum,
has utility in treating Huntington's disease.
[0131] Accordingly, certain aspects of the invention relate to
methods for treating Huntington's disease in a mammal comprising
administering a rAAV particle to the striatum, wherein the rAAV
particle comprises a rAAV vector encoding a heterologous nucleic
acid that is expressed in at least the cerebral cortex and striatum
of the mammal. HD is characterized by progressive symptoms related
to overall movement and motor control, cognition, and mental
health. While the precise nature and extent of symptoms vary
between individuals, symptoms generally progress over time. In most
cases, symptoms begin to appear between 30 and 40 years of age with
subtle disruptions in motor skills, cognition, and personality.
Over time, these progress into jerky, uncontrollable movements and
loss of muscle control, dementia, and psychiatric illnesses such as
depression, aggression, anxiety, and obsessive-compulsive
behaviors. Death typically occurs 10-15 years after the onset of
symptoms. Less than 10% of HD cases involve a juvenile-onset form
of the disease, characterized by a faster disease progression. It
is thought that approximately 1 in 10,000 Americans has HD.
[0132] Most cases of HD are associated with a trinucleotide CAG
repeat expansion in the HTT gene. The number of CAG repeats in the
HTT gene is strongly correlated with the manifestation of HD. For
example, individuals with 35 or fewer repeats typically do not
develop HD, but individuals with between 27 and 35 repeats have a
greater risk of having offspring with HD. Individuals with between
36 and 40-42 repeats have an incomplete penetrance of HD, whereas
individuals with more than 40-42 repeats show complete penetrance.
Cases of juvenile-onset HD may be associated with CAG repeat sizes
of 60 or more.
[0133] The polyQ-expanded Htt protein resulting from this CAG
repeat expansion is associated with cellular aggregates or
inclusion bodies, perturbations to protein homeostasis, and
transcriptional dysregulation. While these toxic phenotypes may be
associated with several parts of the body, they are most typically
associated with neuronal cell death. HD patients often display
cortical thinning and a striking, progressive loss of striatal
neurons. The striatum appears to be the most vulnerable region of
the brain to HD (particularly the striatal medium spiny neurons),
with early effects seen in the putamen and caudate nucleus. Cell
death in the striatal spiny neurons, increased numbers of
astrocytes, and activation of microglia are observed in the brains
of HD patients. HD may also affect certain regions of the
hippocampus, cerebral cortex, thalamus, hypothalamus, and
cerebellum.
[0134] Animal models of HD may be used to test potential
therapeutic strategies, such as the compositions and methods of the
present disclosure. Mouse models for HD are known in the art. These
include mouse models with fragments of mutant HTT such as the R6/1
and N171-82Q HD mice (Harper et al., (2005) Proc. Natl. Acad. Sci.
USA 102:5820-5825, Rodriguez-Lebron et al., (2005) Mol. Ther.
12:618-633, Machida et al., (2006) Biochem. Biophys. Res. Commun.
343:190-197). Another example of a mouse HD model described herein
is the YAC128 mouse model. This model bears a yeast artificial
chromosome (YAC) expressing a mutant human HTT gene with 128 CAG
repeats, and YAC128 mice exhibit significant and widespread
accumulation of Htt aggregates in the striatum by 12 months of age
(Slow et al., (2003) Hum. Mol. Genet. 12:1555-1567, Pouladi et al.,
(2012) Hum. Mol. Genet. 21:2219-2232).
[0135] Other animal models for HD may also be used. For example,
transgenic rat (von Horsten, S. et al. (2003) Hum. Mol. Genet.
12:617-24) and rhesus monkey (Yang, S. H. et al. (2008) Nature
453:921-4) models have been described. Non-genetic models are also
known. These most often involve the use of excitotoxic compounds
(such as quinolinic acid or kainic acid) or mitochondrial toxins
(such as 3-nitropropionic acid and malonic acid) to induce striatal
neuron cell death in rodents or non-human primates (for more
description and references, see Ramaswamy, S. et al. (2007) ILAR J.
48:356-73).
[0136] In some aspects, the invention provides methods for
ameliorating a symptom of HD, comprising administration of a rAAV
particle comprising a rAAV vector encoding a heterologous nucleic
acid that is expressed in at least the cerebral cortex and striatum
to the striatum. In some embodiments, the symptoms of HD include,
but are not limited to, chorea, rigidity, uncontrollable body
movements, loss of muscle control, lack of coordination,
restlessness, slowed eye movements, abnormal posturing,
instability, ataxic gait, abnormal facial expression, speech
problems, difficulties chewing and/or swallowing, disturbance of
sleep, seizures, dementia, cognitive deficits (e.g., diminished
abilities related to planning, abstract thought, flexibility, rule
acquisition, interpersonal sensitivity, self-control, attention,
learning, and memory), depression, anxiety, changes in personality,
aggression, compulsive behavior, obsessive-compulsive behavior,
hypersexuality, psychosis, apathy, irritability, suicidal thoughts,
weight loss, muscle atrophy, heart failure, reduced glucose
tolerance, testicular atrophy, and osteoporosis.
[0137] In some aspects, the invention provides methods to prevent
or delay progression of HD. Autosomal dominant HD is a genetic
disease that can be genotyped. For example, the number of CAG
repeats in HTT may be determined by PCR-based repeat sizing. This
type of diagnosis may be performed at any stage of life through
directly testing juveniles or adults (e.g., along with presentation
of clinical symptoms), prenatal screening or prenatal exclusion
testing (e.g., by chorionic villus sampling or amniocentesis), or
preimplantation screening of embryos. Additionally, HD may be
diagnosed by brain imaging, looking for shrinkage of the caudate
nuclei and/or putamen and/or enlarged ventricles. These symptoms,
combined with a family history of HD and/or clinical symptoms, may
indicate HD.
[0138] Means for determining amelioration of the symptoms of HD are
known in the art. For example, the Unified Huntington's Disease
Rating Scale (UHDRS) may be used to assess motor function,
cognitive function, behavioral abnormalities, and functional
capacity (see, e.g., Huntington Study Group (1996) Movement
Disorders 11:136-42). This rating scale was developed to provide a
uniform, comprehensive test for multiple facets of the disease
pathology, incorporating elements from tests such as the HD
Activities and Daily Living Scale, Marsden and Quinn's chorea
severity scale, the Physical Disability and Independence scales,
the HD motor rating scale (HDMRS), the HD functional capacity scale
(HDFCS), and the quantitated neurological exam (QNE). Other test
useful for determining amelioration of HD symptoms may include
without limitation the Montreal Cognitive Assessment, brain imaging
(e.g., MRI), Category Fluency Test, Trail Making Test, Map Search,
Stroop Word Reading Test, Speeded Tapping Task, and the Symbol
Digit Modalities Test.
[0139] In some aspects of the invention, the methods are used for
the treatment of humans with HD. As described above, HD is
inherited in an autosomal dominant manner and caused by CAG repeat
expansion in the HTT gene. rAAV particles may include, e.g., a
heterologous nucleic acid encoding a therapeutic polypeptide or
nucleic acid that targets HTT. Juvenile-onset HD is most often
inherited from the paternal side. Huntington disease-like
phenotypes have also been correlated with other genetic loci, such
as HDL1, PRNP, HDL2, HDL3, and HDL4. It is thought that other
genetic loci may modify the manifestation of HD symptoms, including
mutations in the GRIN2A, GRIN2B, MSX1, GRIK2, and APOE genes.
[0140] In some embodiments, delivery of recombinant viral particles
is by injection of viral particles to the striatum. Intrastriatal
administration delivers recombinant viral particles to an area of
the brain, the striatum (including the putamen and caudate
nucleus), that is highly affected by HD. In addition, and without
wishing to be bound to theory, it is thought that recombinant viral
particles (e.g., rAAV particles) injected into the striatum may be
also dispersed (e.g., through retrograde transport) to other areas
of the brain, including without limitation projection areas (e.g.,
the cerebral cortex). In some embodiments, the recombinant viral
particles are delivered by convection enhanced delivery (e.g.,
convection enhanced delivery to the striatum).
[0141] In some embodiments, the transgene (e.g., a heterologous
nucleic acid described herein) is operably linked to a promoter.
Exemplary promoters include, but are not limited to, the
cytomegalovirus (CMV) immediate early promoter, the GUSB promoter,
the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK)
promoter, a simian virus 40 (SV40) promoter and a CK6 promoter, a
transthyretin promoter (TTR), a TK promoter, a tetracycline
responsive promoter (TRE), an HBV promoter, an hAAT promoter, a LSP
promoter, chimeric liver-specific promoters (LSPs), the E2F
promoter, the telomerase (hTERT) promoter; the cytomegalovirus
enhancer/chicken beta-actin/Rabbit .beta.-globin promoter (CAG
promoter; Niwa et al., Gene, 1991, 108(2):193-9) and the elongation
factor 1-alpha promoter (EF1-alpha) promoter (Kim et al., Gene,
1990, 91(2):217-23 and Guo et al., Gene Ther., 1996, 3(9):802-10).
In some embodiments, the promoter comprises a human
.beta.-glucuronidase promoter or a cytomegalovirus enhancer linked
to a chicken .beta.-actin (CBA) promoter. The promoter can be a
constitutive, inducible or repressible promoter. In some
embodiments, the invention provides a recombinant vector comprising
nucleic acid encoding a heterologous nucleic acid of the present
disclosure operably linked to a CBA promoter. In some embodiments,
the promoter is a CBA promoter, a minimum CBA promoter, a CMV
promoter or a GUSB promoter.
[0142] Examples of constitutive promoters include, without
limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter
(optionally with the RSV enhancer), the cytomegalovirus (CMV)
promoter (optionally with the CMV enhancer) [see, e.g., Boshart et
al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate
reductase promoter, the 13-actin promoter, the phosphoglycerol
kinase (PGK) promoter, and the EFla promoter [Invitrogen].
[0143] Inducible promoters allow regulation of gene expression and
can be regulated by exogenously supplied compounds, environmental
factors such as temperature, or the presence of a specific
physiological state, e.g., acute phase, a particular
differentiation state of the cell, or in replicating cells only.
Inducible promoters and inducible systems are available from a
variety of commercial sources, including, without limitation,
Invitrogen, Clontech and Ariad. Many other systems have been
described and can be readily selected by one of skill in the art.
Examples of inducible promoters regulated by exogenously supplied
promoters include the zinc-inducible sheep metallothionine (MT)
promoter, the dexamethasone (Dex)-inducible mouse mammary tumor
virus (MMTV) promoter, the T7 polymerase promoter system (WO
98/10088); the ecdysone insect promoter (No et al., Proc. Natl.
Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible
system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551
(1992)), the tetracycline-inducible system (Gossen et al., Science,
268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem.
Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al.,
Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther.,
4:432-441 (1997)) and the rapamycin-inducible system (Magari et
al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of
inducible promoters which may be useful in this context are those
which are regulated by a specific physiological state, e.g.,
temperature, acute phase, a particular differentiation state of the
cell, or in replicating cells only.
[0144] In another embodiment, the native promoter, or fragment
thereof, for the transgene will be used. The native promoter may be
preferred when it is desired that expression of the transgene
should mimic the native expression. The native promoter may be used
when expression of the transgene must be regulated temporally or
developmentally, or in a tissue-specific manner, or in response to
specific transcriptional stimuli. In a further embodiment, other
native expression control elements, such as enhancer elements,
polyadenylation sites or Kozak consensus sequences may also be used
to mimic the native expression.
[0145] In some embodiments, the regulatory sequences impart
tissue-specific gene expression capabilities. In some cases, the
tissue-specific regulatory sequences bind tissue-specific
transcription factors that induce transcription in a tissue
specific manner. Such tissue-specific regulatory sequences (e.g.,
promoters, enhancers, etc.) are well known in the art. Exemplary
tissue-specific regulatory sequences include, but are not limited
to the following tissue specific promoters: neuronal such as
neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol.
Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene
promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5
(1991)), and the neuron-specific vgf gene promoter (Piccioli et
al., Neuron, 15:373-84 (1995)). In some embodiments, the
tissue-specific promoter is a promoter of a gene selected from:
neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP),
adenomatous polyposis coli (APC), and ionized calcium-binding
adapter molecule 1 (Iba-1). Other appropriate tissue specific
promoters will be apparent to the skilled artisan. In some
embodiments, the promoter is a chicken Beta-actin promoter.
[0146] In some embodiments, the promoter expresses the heterologous
nucleic acid in a cell of the CNS. As such, in some embodiments, a
therapeutic polypeptide or a therapeutic nucleic acid of the
invention may be used to treat a disorder of the CNS. In some
embodiments, the promoter expresses the heterologous nucleic acid
in a brain cell. A brain cell may refer to any brain cell known in
the art, including without limitation a neuron (such as a sensory
neuron, motor neuron, interneuron, dopaminergic neuron, medium
spiny neuron, cholinergic neuron, GABAergic neuron, pyramidal
neuron, etc.), a glial cell (such as microglia, macroglia,
astrocytes, oligodendrocytes, ependymal cells, radial glia, etc.),
a brain parenchyma cell, microglial cell, ependymal cell, and/or a
Purkinje cell. In some embodiments, the promoter expresses the
heterologous nucleic acid in a neuron and/or glial cell. In some
embodiments, the neuron is a medium spiny neuron of the caudate
nucleus, a medium spiny neuron of the putamen, a neuron of the
cortex layer IV and/or a neuron of the cortex layer V.
[0147] Various promoters that express transcripts (e.g., a
heterologous transgene) in CNS cells, brain cells, neurons, and
glial cells are known in the art and described herein. Such
promoters can comprise control sequences normally associated with
the selected gene or heterologous control sequences. Often, useful
heterologous control sequences include those derived from sequences
encoding mammalian or viral genes. Examples include, without
limitation, the SV40 early promoter, mouse mammary tumor virus LTR
promoter, adenovirus major late promoter (Ad MLP), a herpes simplex
virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the
CMV immediate early promoter region (CMVIE), a rous sarcoma virus
(RSV) promoter, synthetic promoters, hybrid promoters, and the
like. In addition, sequences derived from nonviral genes, such as
the murine metallothionein gene, may also be used. Such promoter
sequences are commercially available from, e.g., Stratagene (San
Diego, Calif.). CNS-specific promoters and inducible promoters may
be used. Examples of CNS-specific promoters include without
limitation those isolated from CNS-specific genes such as myelin
basic protein (MBP), glial fibrillary acid protein (GFAP), and
neuron specific enolase (NSE). Examples of inducible promoters
include DNA responsive elements for ecdysone, tetracycline,
metallothionein, and hypoxia, inter alia.
[0148] The present invention contemplates the use of a recombinant
viral genome for introduction of one or more nucleic acid sequences
encoding for a heterologous nucleic acid or packaging into an AAV
viral particle. The recombinant viral genome may include any
element to establish the expression of a heterologous transgene,
for example, a promoter, a heterologous nucleic acid, an ITR, a
ribosome binding element, terminator, enhancer, selection marker,
intron, polyA signal, and/or origin of replication. In some
embodiments, the rAAV vector comprises one or more of an enhancer,
a splice donor/splice acceptor pair, a matrix attachment site, or a
polyadenylation signal.
[0149] In some embodiments, the administration of an effective
amount of rAAV particles comprising a vector encoding a therapeutic
nucleic acid or polypeptide transduces cells (e.g., CNS cells,
brain cells, neurons, and/or glial cells) at or near the site of
administration (e.g., the striatum and/or cortex) or more distal to
the site of administration. In some embodiments, more than about
any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75% or 100% of neurons are transduced. In some
embodiments, about 5% to about 100%, about 10% to about 50%, about
10% to about 30%, about 25% to about 75%, about 25% to about 50%,
or about 30% to about 50% of the neurons are transduced. Methods to
identify neurons transduced by recombinant viral particles
expressing miRNA are known in the art; for example,
immunohistochemistry, RNA detection (e.g., qPCR, Northern blotting,
RNA-seq, in situ hybridization, and the like) or the use of a
co-expressed marker such as enhanced green fluorescent protein can
be used to detect expression.
[0150] In some aspects, the invention provides viral particles
comprising a recombinant self-complementing genome (e.g., a
self-complementary rAAV vector). AAV viral particles with
self-complementing vector genomes and methods of use of
self-complementing AAV genomes are described in U.S. Pat. Nos.
6,596,535; 7,125,717; 7,465,583; 7,785,888; 7,790,154; 7,846,729;
8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther
10:2105-2111, each of which are incorporated herein by reference in
its entirety. A rAAV comprising a self-complementing genome will
quickly form a double stranded DNA molecule by virtue of its
partially complementing sequences (e.g., complementing coding and
non-coding strands of a heterologous nucleic acid). In some
embodiments, the vector comprises first nucleic acid sequence
encoding the heterologous nucleic acid and a second nucleic acid
sequence encoding a complement of the nucleic acid, where the first
nucleic acid sequence can form intrastrand base pairs with the
second nucleic acid sequence along most or all of its length.
[0151] In some embodiments, the first heterologous nucleic acid
sequence and a second heterologous nucleic acid sequence are linked
by a mutated ITR (e.g., the right ITR). In some embodiments, the
ITR comprises the polynucleotide sequence
5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC
GGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO:1). The
mutated ITR comprises a deletion of the D region comprising the
terminal resolution sequence. As a result, on replicating an AAV
viral genome, the rep proteins will not cleave the viral genome at
the mutated ITR and as such, a recombinant viral genome comprising
the following in 5' to 3' order will be packaged in a viral capsid:
an AAV ITR, the first heterologous polynucleotide sequence
including regulatory sequences, the mutated AAV ITR, the second
heterologous polynucleotide in reverse orientation to the first
heterologous polynucleotide and a third AAV ITR.
V. Viral Particles and Methods of Producing Viral Particles
[0152] rAAV Viral Particles
[0153] The invention provides methods and systems for administering
rAAV particles. In some embodiments, the rAAV particle comprises a
rAAV vector. In some embodiments, the viral particle is a
recombinant AAV particle comprising a nucleic acid comprising a
heterologous nucleic acid flanked by one or two AAV inverted
terminal repeats (ITRs). The nucleic acid is encapsidated in the
AAV particle. The AAV particle also comprises capsid proteins. In
some embodiments, the nucleic acid comprises the coding sequence(s)
of interest (e.g., a heterologous nucleic acid) operatively linked
components in the direction of transcription, control sequences
including transcription initiation and termination sequences,
thereby forming an expression cassette. The expression cassette is
flanked on the 5' and 3' end by at least one functional AAV ITR
sequence. By "functional AAV ITR sequence" it is meant that the ITR
sequence functions as intended for the rescue, replication and
packaging of the AAV virion. See Davidson et al., PNAS, 2000,
97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and
Pechan et al., Gene Ther., 2009, 16:10-16, all of which are
incorporated herein in their entirety by reference. For practicing
some aspects of the invention, the recombinant vectors comprise at
least all of the sequences of AAV essential for encapsidation and
the physical structures for infection by the rAAV. AAV ITRs for use
in the vectors of the invention need not have a wild-type
nucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther.,
1994, 5:793-801), and may be altered by the insertion, deletion or
substitution of nucleotides or the AAV ITRs may be derived from any
of several AAV serotypes. More than 40 serotypes of AAV are
currently known, and new serotypes and variants of existing
serotypes continue to be identified. See Gao et al., PNAS, 2002,
99(18): 11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis
et al., J. Virol., 2003, 77(12):6799-810. Use of any AAV serotype
is considered within the scope of the present invention. In some
embodiments, a rAAV vector is a vector derived from an AAV
serotype, including without limitation, AAV ITRs are AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10,
AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV,
or mouse AAV or the like. In some embodiments, the nucleic acid in
the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV
DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs or the like.
In certain embodiments, the nucleic acid in the AAV comprises an
AAV2 ITR.
[0154] In some embodiments, a vector may include a stuffer nucleic
acid. In some embodiments, the stuffer nucleic acid may encode a
green fluorescent protein. In some embodiments, the stuffer nucleic
acid may be located between the promoter and the nucleic acid
encoding the RNAi.
[0155] In further embodiments, the rAAV particles comprise an AAV1
capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5
capsid, an AAV6 capsid (e.g., a wild-type AAV6 capsid, or a variant
AAV6 capsid such as ShH10, as described in U.S. PG Pub.
2012/0164106), an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an
AAVrh8R capsid, an AAV9 capsid (e.g., a wild-type AAV9 capsid, or a
modified AAV9 capsid as described in U.S. PG Pub. 2013/0323226), an
AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid,
a tyrosine capsid mutant, a heparin binding capsid mutant, an
AAV2R471A capsid, an AAVAAV2/2-7m8 capsid, an AAV DJ capsid (e.g.,
an AAV-DJ/8 capsid, an AAV-DJ/9 capsid, or any other of the capsids
described in U.S. PG Pub. 2012/0066783), an AAV2 N587A capsid, an
AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a
goat AAV capsid, an AAV1/AAV2 chimeric capsid, a bovine AAV capsid,
a mouse AAV capsid, a rAAV2/HBoV1 capsid, or an AAV capsid
described in U.S. Pat. No. 8,283,151 or International Publication
No. WO/2003/042397. In some embodiments, a mutant capsid protein
maintains the ability to form an AAV capsid. In some embodiments,
the rAAV particle comprises AAV5 tyrosine mutant capsid (Zhong L.
et al., (2008) Proc Natl Acad Sci USA 105(22):7827-7832. In further
embodiments, the rAAV particle comprises capsid proteins of an AAV
serotype from Clades A-F (Gao, et al., J. Virol. 2004,
78(12):6381). In some embodiments, the rAAV particle comprises an
AAV1 capsid protein or mutant thereof. In other embodiments, the
rAAV particle comprises an AAV2 capsid protein or mutant thereof.
In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6,
AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some
embodiments, the rAAV particle comprises an AAV serotype 1 (AAV1)
capsid. In some embodiments, the rAAV particle comprises an AAV
serotype 2 (AAV2) capsid.
[0156] Different AAV serotypes are used to optimize transduction of
particular target cells or to target specific cell types within a
particular target tissue (e.g., a CNS tissue). A rAAV particle can
comprise viral proteins and viral nucleic acids derived from the
same serotype or different serotypes (e.g., a mixed serotype). For
example, in some embodiments a rAAV particle can comprise AAV1
capsid proteins and at least one AAV2 ITR or it can comprise AAV2
capsid proteins and at least one AAV1 ITR. Any combination of AAV
serotypes for production of a rAAV particle is provided herein as
if each combination had been expressly stated herein. In some
embodiments, the invention provides rAAV particles comprising an
AAV1 capsid and a rAAV vector of the present disclosure (e.g., an
expression cassette comprising a heterologous nucleic acid),
flanked by at least one AAV2 ITR. In some embodiments, the
invention provides rAAV particles comprising an AAV2 capsid. In
some embodiments, the ITR and the capsid are derived from AAV2. In
some embodiments, the ITR is derived from AAV2 and the capsid is
derived from AAV1.
[0157] Production of AAV Particles
[0158] Numerous methods are known in the art for production of rAAV
vectors, including transfection, stable cell line production, and
infectious hybrid virus production systems which include
adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, J E et
al., (1997) J. Virology 71(11):8780-8789) and baculovirus-AAV
hybrids. rAAV production cultures for the production of rAAV virus
particles all require; 1) suitable host cells, including, for
example, human-derived cell lines such as HeLa, A549, or 293 cells,
or insect-derived cell lines such as SF-9, in the case of
baculovirus production systems; 2) suitable helper virus function,
provided by wild-type or mutant adenovirus (such as temperature
sensitive adenovirus), herpes virus, baculovirus, or a plasmid
construct providing helper functions; 3) AAV rep and cap genes and
gene products; 4) a nucleic acid (such as a therapeutic nucleic
acid) flanked by at least one AAV ITR sequences; and 5) suitable
media and media components to support rAAV production. In some
embodiments, the AAV rep and cap gene products may be from any AAV
serotype. In general, but not obligatory, the AAV rep gene product
is of the same serotype as the ITRs of the rAAV vector genome as
long as the rep gene products may function to replicated and
package the rAAV genome. Suitable media known in the art may be
used for the production of rAAV vectors. These media include,
without limitation, media produced by Hyclone Laboratories and JRH
including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle
Medium (DMEM), custom formulations such as those described in U.S.
Pat. No. 6,566,118, and Sf-900 II SFM media as described in U.S.
Pat. No. 6,723,551, each of which is incorporated herein by
reference in its entirety, particularly with respect to custom
media formulations for use in production of recombinant AAV
vectors. In some embodiments, the AAV helper functions are provided
by adenovirus or HSV. In some embodiments, the AAV helper functions
are provided by baculovirus and the host cell is an insect cell
(e.g., Spodoptera frugiperda (Sf9) cells).
[0159] In some embodiments, rAAV particles may be produced by a
triple transfection method, such as the exemplary triple
transfection method provided infra. Briefly, a plasmid containing a
rep gene and a capsid gene, along with a helper adenoviral plasmid,
may be transfected (e.g., using the calcium phosphate method) into
a cell line (e.g., HEK-293 cells), and virus may be collected and
optionally purified. As such, in some embodiments, the rAAV
particle was produced by triple transfection of a nucleic acid
encoding the rAAV vector, a nucleic acid encoding AAV rep and cap,
and a nucleic acid encoding AAV helper virus functions into a host
cell, wherein the transfection of the nucleic acids to the host
cells generates a host cell capable of producing rAAV
particles.
[0160] In some embodiments, rAAV particles may be produced by a
producer cell line method, such as the exemplary producer cell line
method provided infra (see also (referenced in Martin et al.,
(2013) Human Gene Therapy Methods 24:253-269). Briefly, a cell line
(e.g., a HeLa cell line) may be stably transfected with a plasmid
containing a rep gene, a capsid gene, and a promoter-heterologous
nucleic acid sequence. Cell lines may be screened to select a lead
clone for rAAV production, which may then be expanded to a
production bioreactor and infected with an adenovirus (e.g., a
wild-type adenovirus) as helper to initiate rAAV production. Virus
may subsequently be harvested, adenovirus may be inactivated (e.g.,
by heat) and/or removed, and the rAAV particles may be purified. As
such, in some embodiments, the rAAV particle was produced by a
producer cell line comprising one or more of nucleic acid encoding
the rAAV vector, a nucleic acid encoding AAV rep and cap, and a
nucleic acid encoding AAV helper virus functions.
[0161] In some aspects, a method is provided for producing any rAAV
particle as disclosed herein comprising (a) culturing a host cell
under a condition that rAAV particles are produced, wherein the
host cell comprises (i) one or more AAV package genes, wherein each
said AAV packaging gene encodes an AAV replication and/or
encapsidation protein; (ii) a rAAV pro-vector comprising a nucleic
acid encoding a heterologous nucleic acid as described herein
flanked by at least one AAV ITR, and (iii) an AAV helper function;
and (b) recovering the rAAV particles produced by the host cell. In
some embodiments, said at least one AAV ITR is selected from the
group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV
DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs or the like.
In some embodiments, said encapsidation protein is selected from
the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,
AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12,
AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A,
AAV V708K, goat AAV, AAV1/AAV2 chimeric, bovine AAV, or mouse AAV
capsid rAAV2/HBoV1 serotype capsid proteins or mutants thereof. In
some embodiments, the encapsidation protein is an AAV5 capsid
protein including AAV5 capsid proteins having tyrosine capsid
mutations. In some embodiments, the encapsidation protein is an
AAV5 capsid protein including AAV5 capsid proteins having tyrosine
capsid mutations and the ITR is an AAV2 ITR. In further
embodiments, the rAAV particle comprises capsid proteins of an AAV
serotype from Clades A-F. In some embodiments, the rAAV particles
comprise an AAV1 capsid and a recombinant genome comprising AAV2
ITRs, a mutant AAV2 ITR and nucleic acid encoding a therapeutic
transgene/nucleic acid. In some embodiments, the AAV ITRs are AAV
ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV5, AAVrh8,
AAVrh8R, AAV5, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a
goat AAV, bovine AAV, or mouse AAV serotype ITRs. In certain
embodiments, the AAV ITRs are AAV2 ITRs.
[0162] Suitable rAAV production culture media of the present
invention may be supplemented with serum or serum-derived
recombinant proteins at a level of 0.5%-20% (v/v or w/v).
Alternatively, as is known in the art, rAAV vectors may be produced
in serum-free conditions which may also be referred to as media
with no animal-derived products. One of ordinary skill in the art
may appreciate that commercial or custom media designed to support
production of rAAV vectors may also be supplemented with one or
more cell culture components know in the art, including without
limitation glucose, vitamins, amino acids, and or growth factors,
in order to increase the titer of rAAV in production cultures.
[0163] rAAV production cultures can be grown under a variety of
conditions (over a wide temperature range, for varying lengths of
time, and the like) suitable to the particular host cell being
utilized. As is known in the art, rAAV production cultures include
attachment-dependent cultures which can be cultured in suitable
attachment-dependent vessels such as, for example, roller bottles,
hollow fiber filters, microcarriers, and packed-bed or
fluidized-bed bioreactors. rAAV vector production cultures may also
include suspension-adapted host cells such as HeLa, 293, and SF-9
cells which can be cultured in a variety of ways including, for
example, spinner flasks, stirred tank bioreactors, and disposable
systems such as the Wave bag system.
[0164] rAAV vector particles of the invention may be harvested from
rAAV production cultures by lysis of the host cells of the
production culture or by harvest of the spent media from the
production culture, provided the cells are cultured under
conditions known in the art to cause release of rAAV particles into
the media from intact cells, as described more fully in U.S. Pat.
No. 6,566,118). Suitable methods of lysing cells are also known in
the art and include for example multiple freeze/thaw cycles,
sonication, microfluidization, and treatment with chemicals, such
as detergents and/or proteases.
[0165] In a further embodiment, the rAAV particles are purified.
The term "purified" as used herein includes a preparation of rAAV
particles devoid of at least some of the other components that may
also be present where the rAAV particles naturally occur or are
initially prepared from. Thus, for example, isolated rAAV particles
may be prepared using a purification technique to enrich it from a
source mixture, such as a culture lysate or production culture
supernatant. Enrichment can be measured in a variety of ways, such
as, for example, by the proportion of DNase-resistant particles
(DRPs) or genome copies (gc) present in a solution, or by
infectivity, or it can be measured in relation to a second,
potentially interfering substance present in the source mixture,
such as contaminants, including production culture contaminants or
in-process contaminants, including helper virus, media components,
and the like.
[0166] In some embodiments, the rAAV production culture harvest is
clarified to remove host cell debris. In some embodiments, the
production culture harvest is clarified by filtration through a
series of depth filters including, for example, a grade DOHC
Millipore Millistak+HC Pod Filter, a grade A1HC Millipore
Millistak+HC Pod Filter, and a 0.2 .mu.m Filter Opticap XL1O
Millipore Express SHC Hydrophilic Membrane filter. Clarification
can also be achieved by a variety of other standard techniques
known in the art, such as, centrifugation or filtration through any
cellulose acetate filter of 0.2 .mu.m or greater pore size known in
the art.
[0167] In some embodiments, the rAAV production culture harvest is
further treated with Benzonase.RTM. to digest any high molecular
weight DNA present in the production culture. In some embodiments,
the Benzonase.RTM. digestion is performed under standard conditions
known in the art including, for example, a final concentration of
1-2.5 units/ml of Benzonase.RTM. at a temperature ranging from
ambient to 37.degree. C. for a period of 30 minutes to several
hours.
[0168] rAAV particles may be isolated or purified using one or more
of the following purification steps: equilibrium centrifugation;
flow-through anionic exchange filtration; tangential flow
filtration (TFF) for concentrating the rAAV particles; rAAV capture
by apatite chromatography; heat inactivation of helper virus; rAAV
capture by hydrophobic interaction chromatography; buffer exchange
by size exclusion chromatography (SEC); nanofiltration; and rAAV
capture by anionic exchange chromatography, cationic exchange
chromatography, or affinity chromatography. These steps may be used
alone, in various combinations, or in different orders. In some
embodiments, the method comprises all the steps in the order as
described below. Methods to purify rAAV particles are found, for
example, in Xiao et al., (1998) Journal of Virology 72:2224-2232;
U.S. Pat. Nos. 6,989,264 and 8,137,948; and WO 2010/148143.
[0169] In some embodiments, the rAAV particle is in a
pharmaceutical composition. The pharmaceutical compositions may be
suitable for any mode of administration described herein. A
pharmaceutical composition of a recombinant viral particle
comprising a nucleic acid encoding a therapeutic transgene/nucleic
acid can be introduced to the CNS (e.g., the striatum and/or
cerebral cortex).
[0170] In some embodiments, the rAAV particle is in a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient. As is well known in the art, pharmaceutically acceptable
excipients are relatively inert substances that facilitate
administration of a pharmacologically effective substance and can
be supplied as liquid solutions or suspensions, as emulsions, or as
solid forms suitable for dissolution or suspension in liquid prior
to use. For example, an excipient can give form or consistency, or
act as a diluent. Suitable excipients include but are not limited
to stabilizing agents, wetting and emulsifying agents, salts for
varying osmolarity, encapsulating agents, pH buffering substances,
and buffers. Such excipients include any pharmaceutical agent
suitable for direct delivery to the eye which may be administered
without undue toxicity. Pharmaceutically acceptable excipients
include, but are not limited to, sorbitol, any of the various TWEEN
compounds, and liquids such as water, saline, glycerol and ethanol.
Pharmaceutically acceptable salts can be included therein, for
example, mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the like.
A thorough discussion of pharmaceutically acceptable excipients is
available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co.,
N.J. 1991).
[0171] In some embodiments, the administered composition includes
rAAV particles and poloxamer. The term "poloxamer" may encompass
many compounds because different lengths for the polyoxypropylene
and polyoxyethylene chains may be used in combination. For example,
a poloxamer may have the chemical formula of
HO(C.sub.2H.sub.4O).sub.n(C.sub.3H.sub.6O).sub.m(C.sub.2H.sub.4O).sub.nH,
where n (i.e., the polyoxyethylene chain length) has a value from
about 60 to about 150, and m (i.e., the polyoxypropylene chain
length) has a value from about 25 to about 60.
[0172] In some embodiments, the poloxamer is poloxamer 188 (e.g.,
CAS No. 9003-11-6). Poloxamers may be described by a numbering
system that designates their approximate molecular weight and
percentage of polyoxyethylene content. These values often refer to
an average value in a poloxamer composition, rather than an
absolute value of each poloxamer molecule in the composition. Under
this methodology, the first two digits are multiplied by 100 to
give the approximate molecular weight of the polyoxypropylene
block, and the third digit is multiplied by 10 to give the
percentage by weight of the polyoxyethylene block. For example,
poloxamer 188 may refer to a poloxamer with n having a value of
about 80 and with m having a value of about 27 as in the formula
depicted above. Poloxamer 188 may have an average molecular weight
of from about 7680 to about 9510 g/mol.
[0173] Poloxamers sold under a trade name such as PLURONIC.RTM. may
be named under a different methodology. A letter may be used to
indicate the physical state (e.g., F for solid, P for paste, or L
for liquid). A 2 or 3 digit number may be used to indicate the
chemical properties. The first one or two digits are multiplied by
300 to give the approximate molecular weight of the
polyoxypropylene block, and the third digit is multiplied by 10 to
give the percentage by weight of the polyoxyethylene block. For
example, PLURONIC.RTM. or LUTROL.RTM. F68 may refer to a solid
poloxamer with n having a value of about 80 and with m having a
value of about 27 as in the formula depicted above. Therefore, in
some embodiments, the poloxamer 188 may be PLURONIC.RTM. F68 or
LUTROL.RTM. F68.
[0174] In some embodiments, the concentration of poloxamer in the
composition ranges from about 0.0001% to about 0.01%. In some
embodiments, the concentration of poloxamer in the composition is
less than about any of the following percentages: 0.01, 0.005,
0.001, or 0.0005. In some embodiments, the concentration of
poloxamer in the composition is greater than about any of the
following percentages: 0.0001, 0.0005, 0.001, or 0.005. That is,
the concentration of poloxamer in the composition can be any of a
range of percentages having an upper limit of 0.01, 0.005, 0.001,
or 0.0005 and an independently selected lower limit of 0.0001,
0.0005, 0.001, or 0.005, wherein the lower limit is less than the
upper limit. In certain embodiments, the concentration of poloxamer
in the composition is about 0.001%.
[0175] In some embodiments, the composition further comprises
sodium chloride. In some embodiments, the concentration of sodium
chloride in the composition ranges from about 100 mM to about 250
mM. In some embodiments, the concentration of sodium chloride in
the composition is less than about any of the following
concentrations (in mM): 250, 225, 200, 175, 150, or 125. In some
embodiments, the concentration of sodium chloride in the
composition is greater than about any of the following
concentrations (in mM): 100, 125, 150, 175, 200, or 225. That is,
the concentration of sodium chloride in the composition can be any
of a range of concentrations (in mM) having an upper limit of 250,
225, 200, 175, 150, or 125 and an independently selected lower
limit of 100, 125, 150, 175, 200, or 225, wherein the lower limit
is less than the upper limit. In certain embodiments, the
concentration of sodium chloride in the composition is about 180
mM.
[0176] In some embodiments, the composition further comprises
sodium phosphate. Sodium phosphate may refer to any single species
of sodium phosphate (e.g., monobasic sodium phosphate, dibasic
sodium phosphate, tribasic sodium phosphate, and so forth), or it
may refer to sodium phosphate buffer, a mixture of monobasic and
dibasic sodium phosphate solutions. Recipes for sodium phosphate
buffers across a range of pH may be found in a variety of standard
molecular biology protocols, such as the Promega Protocols &
Applications Guide, "Buffers for Biochemical Reactions," Appendix B
part C.
[0177] In some embodiments, the concentration of sodium phosphate
in the composition ranges from about 5 mM to about 20 mM. In some
embodiments, the concentration of sodium phosphate in the
composition is less than about any of the following concentrations
(in mM): 20, 15, or 10. In some embodiments, the concentration of
sodium phosphate in the composition is greater than about any of
the following concentrations (in mM): 5, 10, or 15. That is, the
concentration of sodium phosphate in the composition can be any of
a range of concentrations (in mM) having an upper limit of 20, 15,
or 10 and an independently selected lower limit of 5, 10, or 15,
wherein the lower limit is less than the upper limit. In certain
embodiments, the concentration of sodium phosphate in the
composition is about 10 mM.
[0178] In some embodiments, the pH of sodium phosphate in the
composition is about 7.0 to about 8.0. For example, in some
embodiments, the pH of sodium phosphate in the composition is about
7.0, about 7.2, about 7.4, about 7.5, about 7.6, about 7.8, or
about 8.0. In certain embodiments, the pH of sodium phosphate in
the composition is about 7.5. Any of the pH values for sodium
phosphate described herein may be combined with any of the
concentration values for sodium phosphate described above. For
example, in some embodiments, the concentration of sodium phosphate
in the composition is about 10 mM, and the pH is about 7.5.
[0179] In some embodiments, the pharmaceutical composition
comprising a rAAV particle described herein and a pharmaceutically
acceptable carrier is suitable for administration to human. Such
carriers are well known in the art (see, e.g., Remington's
Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and
1570-1580). In some embodiments, the pharmaceutical composition
further comprises a poloxamer (e.g., poloxamer 188, such as
PLURONIC.RTM. or LUTROL.RTM. F68). In some embodiments, the
pharmaceutical composition comprising a rAAV described herein and a
pharmaceutically acceptable carrier is suitable for injection into
the CNS of a mammal.
[0180] Such pharmaceutically acceptable carriers can be sterile
liquids, such as water and oil, including those of petroleum,
animal, vegetable or synthetic origin, such as peanut oil, soybean
oil, mineral oil, and the like. Saline solutions and aqueous
dextrose, polyethylene glycol (PEG) and glycerol solutions can also
be employed as liquid carriers, particularly for injectable
solutions. The pharmaceutical composition may further comprise
additional ingredients, for example preservatives, buffers,
tonicity agents, antioxidants and stabilizers, nonionic wetting or
clarifying agents, viscosity-increasing agents, and the like. The
pharmaceutical compositions described herein can be packaged in
single unit dosages or in multidosage forms. The compositions are
generally formulated as sterile and substantially isotonic
solution.
VI. Systems for Delivery of rAAV Particles
[0181] Also provided are systems for expression of a heterologous
nucleic acid in the cerebral cortex and striatum of a mammal,
comprising (a) a composition comprising rAAV particles, wherein the
rAAV particles comprise a rAAV vector encoding the heterologous
nucleic acid; and (b) a device for delivery of the rAAV particles
to the striatum. The systems and devices of the invention may be
used to deliver any of the rAAV particles described herein to the
CNS (e.g., the striatum) of a mammal. As described above, a rAAV
particle delivered to the striatum may be used to introduce a rAAV
vector encoding a heterologous nucleic acid for expression in the
cerebral cortex and striatum.
[0182] In some embodiments, the rAAV particle is delivered by
convection enhanced delivery (CED). CED is based on pumping an
infusate (e.g., a composition containing a rAAV particle) into the
CNS under pressure in which the hydrostatic pressure of the
interstitial fluid is overcome. This brings the infusate into
contact with the CNS perivasculature, which is utilized like a pump
to distribute the infusate through convection and enhance the
extent of its delivery (see, e.g., Hadaczek et al., (2006) Hum.
Gene Ther. 17:291-302; Bankiewicz et al., (2000) Exp. Neurol.
164:2-14; Sanftner, L M et al., (2005) Exp. Neurol. 194(2):476-483;
Forsayeth, J R et al., (2006)Mol. Ther. 14(4):571-577; U.S. Pat.
No. 6,953,575; U.S. Pat. App. Pub. No. 2002/0141980; U.S. Pat. App.
Pub. No. 2007/0259031; WO 99/61066; and WO 2010/088560).
[0183] As described herein, an advantage of using CED is the
enhanced distribution of the infusate throughout the brain. CED may
result in improved delivery at the site of injection within the
brain (e.g., the striatum, caudate nucleus, and/or putamen). In
addition, delivery to other regions of the brain (e.g., the
cerebral cortex, frontal cortex, prefrontal association cortical
areas, premotor cortex, primary somatosensory cortical areas,
and/or primary motor cortex) may be achieved through CED. Without
wishing to be bound to theory, it is also thought that recombinant
viral particles (e.g., rAAV particles) injected into the striatum
may be also dispersed (e.g., through retrograde transport) to other
areas of the brain, including without limitation projection areas
(e.g., the cortex).
[0184] In some embodiments, the rAAV particle is delivered using a
CED delivery system. AAV particles may be delivered by CED (see,
e.g., WO 99/61066). As described herein, CED may be accomplished
using any of the systems described herein. Devices for CED (e.g.,
for delivery of a composition including rAAV particles) are known
in the art and generally employ a pump (e.g., an osmotic and/or
infusion pump, as described below) and an injection device (e.g., a
catheter, cannula, etc.). Optionally, an imaging technique may be
used to guide the injection device and/or monitor delivery of the
infusate (e.g., a composition including rAAV particles). The
injection device may be inserted into the CNS tissue in the
subject. One of skill in the art is able to determined suitable
coordinates for positioning the injection device in the target CNS
tissue. In some embodiments, positioning is accomplished through an
anatomical map obtained for example by CT and/or MRI imaging of the
subject's brain to guide the injection device to the target CNS
tissue. In some embodiments, iMRI and/or real-time imaging of the
delivery may be performed. In some embodiments, the device is used
to administer rAAV particles to a mammal by the methods of the
invention.
[0185] In some embodiments, intraoperative magnetic resonance
imaging (iMRI) and/or real-time imaging of the delivery may be
performed. In some embodiments, the device is used to administer
rAAV particles to a mammal by the methods of the invention. iMRI is
known in the art as a technique for MRI-based imaging of a patient
during surgery, which helps confirm a successful surgical procedure
(e.g., to deliver rAAV particles to the CNS) and reduces the risk
of damaging other parts of the tissue (for further descriptions,
see, e.g., Fiandaca et al., (2009) Neuroimage 47 Suppl. 2:T27-35).
In some embodiments, a tracing agent (e.g., an MRI contrast
enhancing agent) may be co-delivered with the infusate (e.g., a
composition including rAAV particles) to provide for real-time
monitoring of tissue distribution of infusate. See for example
Fiandaca et al., (2009) Neuroimage 47 Suppl. 2:T27-35; U.S. PG Pub
2007/0259031; and U.S. Pat. No. 7,922,999. Use of a tracing agent
may inform the cessation of delivery. Other tracing and imaging
means known in the art may also be used to follow infusate
distribution.
[0186] In some embodiments, rAAV particles may be administered by
standard stereotaxic injection using devices and methods known in
the art for delivery of rAAV particles. Generally, these methods
may use an injection device, a planning system for translating a
region of the tissue targeted for delivery into a series of
coordinates (e.g., parameters along the latero-lateral,
dorso-ventral, and rostro-caudal axes), and a device for
stereotaxic localization according to the planned coordinates (a
stereotactic device, optionally including the probe and a structure
for fixing the head in place in alignment with the coordinate
system). A non-limiting example of a system that may be useful for
MRI-guided surgery and/or stereotaxic injection is the
ClearPoint.RTM. system (MRI Interventions, Memphis, Tenn.).
[0187] In some embodiments, the device for convection enhanced
delivery comprises a pump (e.g., an osmotic pump and/or an infusion
pump). Osmotic and/or infusion pumps are commercially available
(e.g., from ALZET.RTM. Corp., Hamilton Corp., ALZA Inc. in Palo
Alto, Calif.). Pump systems may be implantable. Exemplary pump
systems may be found, e.g., in U.S. Pat. Nos. 7,351,239; 7,341,577;
6,042,579; 5,735,815; and 4,692,147. in some embodiments, the pump
is a manual pump. Exemplary devices for CED, including
reflux-resistant and stepped cannulae, may be found in WO 99/61066
and WO 2006/042090, which are hereby incorporated by reference in
its entirety.
[0188] In some embodiments, the device for convection enhanced
delivery comprises a reflux-resistant cannula (e.g., a reflux-free
step design cannula). Further descriptions and exemplary
reflux-resistant cannulae may be found, for example, in Krauze et
al., (2009) Methods Enzymol. 465:349-362; U.S. PG Pub 2006/0135945;
U.S. PG Pub 2007/0088295; and PCT/US08/64011. In some embodiments,
only one cannula is used. In other embodiments, more than one
cannula is used. In some embodiments, the device for convection
enhanced delivery comprises a reflux-resistant cannula joined with
a pump that produces enough pressure to cause the infusate to flow
through the cannula to the target tissue at controlled rates. Any
suitable flow rate can be used such that the intracranial pressure
is maintained at suitable levels so as not to injure the brain
tissue.
[0189] In some embodiments, the cannula is a stepped cannula. As
described, e.g., in WO 2006/042090, a stepped cannula has a number
of steps (e.g., four in FIG. 1 of WO 2006/042090). The steps
nearest the distal end of the cannula are those that enter the
target tissue first, and, accordingly, the number of steps entering
the target tissue (e.g., the striatum) will depend on the depth of
penetration needed to reach that target in the subject. With
respect to delivery to the brain, the operator can readily
determine the appropriate depth of penetration, taking into account
both the size of the subject being treated and the location within
the brain that is being targeted.
[0190] The cannula may be connected to a pump through a system of
tubing. Tubing extends through the lumen of cannula and the
infusate may be delivered through this tubing. In embodiments
containing the tubing, the tubing may be flush with the distal end
of the cannula. Alternatively, the tubing extends from the distal
end of the cannula, in such embodiments, the amount which the
tubing extends may vary depending on the application. Generally,
the tubing will extend from about 1 mm to about 1 cm from the
cannula (or any length therebetween), e.g., from about 1 to about
50 mm (or any length therebetween), or from about 1 mm to about 25
mm (or any length therebetween, including, but not limited to, 1
mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm,
12 mm, 13 min, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21
mm, mm, 23 mm, 24 mm or 25 mm), such as 10 mm beyond the distal end
thereof.
[0191] The tubing extending through the cannula may have a coating
or surrounding material in one or more regions, for example to
protect the tubing in contact with the infusate. Thus, in certain
embodiments, tubing (e.g., FEP (Teflon) tubing) protects the
portion of the fused silica tubing extending beyond the proximal
end of the stainless steel cannula. The fused silica tubing may be
connected to the syringe by any suitable means, including, but not
limited to, a Luer compression fitting, and the syringe is driven
by a syringe pump (manual, electronic and/or computerized). It will
apparent that the syringe size can be selected by the operator to
deliver the appropriate amount of product(s). Thus, 1 mL, 2.5 mL, 5
mL, or even larger syringes may be used.
[0192] Stepped cannulae may be made out of the variety of materials
that are physiologically acceptable, including without limitation
stainless steel (e.g. 316SS or 304SS), metal, metal alloys,
polymers, organic fibers, inorganic fibers and/or combinations
thereof.
[0193] Optionally, an infusate-contact surface (e.g., tubing or
coating) may extend through the lumen of the cannula. A variety of
materials may also be used for the o infusate-contact surface,
including but not limited to metals, metal alloys, polymers,
organic fibers, inorganic fibers and/or combinations thereof. In
some embodiments, the product-contact surface is not stainless
steel. In such embodiments, the outer cannula may still be made of
a material physiologically compatible with the target tissue, but
there since there is no product contact it need not be compatible
with the biologically active agent or product formulation.
[0194] In some embodiments, penetration of the infusate is further
augmented by the use of a facilitating agent. A facilitating agent
is capable of further facilitating the delivery of infusate to
target tissue (e.g., CNS target tissue). A non-limiting example of
a facilitating agent is low molecular weight heparin (see, e.g.,
U.S. Pat. No. 7,922,999).
[0195] Suitable packaging for pharmaceutical compositions described
herein are known in the art, and include, for example, vials (such
as sealed vials), vessels, ampules, bottles, jars, flexible
packaging (e.g., sealed Mylar or plastic bags), and the like. These
articles of manufacture may further be sterilized and/or
sealed.
[0196] Further provided herein are methods for treating a disorder
of the CNS in a mammal comprising administering a rAAV particle to
the mammal according to the methods described herein. Yet further
provided are methods for treating Huntington's disease in a mammal
comprising administering a rAAV particle to the mammal according to
the methods described herein using a system as described
herein.
[0197] The present invention also provides kits for administering a
rAAV particle described herein to a mammal according to the methods
of the invention. The kits may comprise any of the rAAV particles
or rAAV particle compositions of the invention. For example, the
kits may include rAAV particles with a rAAV vector encoding a
heterologous nucleic acid that is expressed in at least the
cerebral cortex and striatum of a mammal. In some embodiments, the
kits further comprise any of the devices or systems described
above.
[0198] In some embodiments, the kits further include instructions
for CNS delivery (e.g., delivery to the striatum of a mammal) of
the composition of rAAV particles. The kits described herein may
further include other materials desirable from a commercial and
user standpoint, including other buffers, diluents, filters,
needles, syringes, and package inserts with instructions for
performing any methods described herein. Suitable packaging
materials may also be included and may be any packaging materials
known in the art, including, for example, vials (such as sealed
vials), vessels, ampules, bottles, jars, flexible packaging (e.g.,
sealed Mylar or plastic bags), and the like. These articles of
manufacture may further be sterilized and/or sealed. In some
embodiments, the kits comprise instructions for treating a disorder
of the CNS described herein using any of the methods and/or rAAV
particles described herein. The kits may include a pharmaceutically
acceptable carrier suitable for injection into the CNS of an
individual, and one or more of: a buffer, a diluent, a filter, a
needle, a syringe, and a package insert with instructions for
performing injections into the striatum of a mammal.
[0199] In some embodiments, the kits further contain one or more of
the buffers and/or pharmaceutically acceptable excipients described
herein (e.g., as described in REMINGTON'S PHARMACEUTICAL SCIENCES
(Mack Pub. Co., N.J. 1991). In some embodiments, the kits include
one or more pharmaceutically acceptable excipients, carriers,
solutions, and/or additional ingredients described herein. The kits
described herein can be packaged in single unit dosages or in
multidosage forms. The contents of the kits are generally
formulated as sterile and can be lyophilized or provided as a
substantially isotonic solution.
EXAMPLES
[0200] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims.
Example 1: Widespread GFP Expression after Intrastriatal AAV1
Vector Delivery
[0201] The ability of AAV1 to efficiently target both striatal and
cortical structures in the Rhesus monkey brain when delivered via
convection-enhanced delivery (CED) was evaluated. AAV vectors
containing GFP cDNA under the control of cytomegalovirus
enhancer/chicken beta-actin (CBA) promoter were infused into the
caudate and putamen of 9 adult male Rhesus monkeys using CED (see,
e.g., Bankiewicz et al., (2000) Exp. Neurol. 164:2-14 and WO
2010/088560).
[0202] Methods
Surgical Delivery
[0203] Nine adult male Rhesus macaques (Macaca mulatta; 8.9-11.9
kg) were included in this study. All animals received an infusion
of AAV vector bilaterally into caudate nucleus and putamen by means
of MRI-guided CED (Richardson, R. M. et al. (2011) Neurosurgery
69:154-163; Richardson, R. M. et al. (2011) Stereotact. Funct.
Neurosurg. 89:141-151; Richardson, R. M. et al. (2011)Mol. Ther.
19:1048-1057). Immediately prior to surgery, animals were
anesthetized with Ketamine HCL (10 mg/kg), weighed, intubated, and
maintained on 1-5% isoflurane. The head was mounted onto a
stereotaxic frame, and the animal transported to the MRI (Siemens
3.0 T Trio MR unit) for a T1-weighted planning scan. After
scanning, animals were transferred to the operating room and the
head prepared for an implantation procedure, and a ceramic
custom-designed fused silica reflux-resistant cannula with a 3-mm
stepped tip was used for the infusion. Temporary guide cannula were
implanted bilaterally (one per hemisphere) using standard
methods.
[0204] Animals received bilateral infusions into caudate nucleus
and putamen of either AAV1-eGFP or AAV2-eGFP vectors obtained with
2 different production methods: Triple Transfection (TT) or
Producer Cell Line (PCL). Vector concentrations and doses are
described in Table 5. Animals were tested for the presence of
anti-AAV1 and anti-AAV2 neutralizing antibodies as previously
described and were considered seronegative as they presented
antibody titers of <1:32 (Bevan, A. K. et al. (2011) Mol. Ther.
19:1971-1980). Survival time was 1 month after AAV delivery for all
the animals.
[0205] Each animal received up to three microinjections per
hemisphere to target the caudate and the putamen (pre-commissural
and post-commissural) regions, as shown in Table 2. To visualize
infusate distribution during MRI, Prohance (2 mM gadoteridol) was
added to the virus. Approximately 30 .mu.l of AAV was administered
into the caudate and 60 .mu.l into the putamen using the convection
enhanced delivery (CED) method (i.e., 90 .mu.L per hemisphere). The
infusion rate was ramped up to a maximum of 5 .mu.L/min.
TABLE-US-00002 TABLE 2 Parenchymal dose volumes per site. Vector
Dose per Number of Dose Production Hemisphere Subject Target Dosing
Volume Method (vg) Number Structure Hemisphere Sites (.mu.L)
AAV1-eGFP 1.71 .times. 10.sup.11 1 Putamen Right 2 30 + 30 (TT)
Left 2 30 + 30 Caudate Right 1 30 Left 1 30 2 Putamen Right 2 30 +
30 Left 2 30 + 30 Caudate Right 1 30 Left 1 30 3 Putamen Right 2 30
+ 30 Left 2 30 + 30 Caudate Right 1 30 Left 1 30 AAV2-eGFP 1.71
.times. 10.sup.11 6 Putamen Right 2 30 + 30 (TT) Left 1 60 Caudate
Right 1 30 Left 2 15 + 15 7 Putamen Right 1 60 Left 2 30 + 30
Caudate Right 1 30 Left 1 30 AAV1-eGFP 1.71 .times. 10.sup.11 5
Putamen Right 1 60 (PCL) Left 1 60 Caudate Right 1 30 Left 2 18 +
20 Left: 4 Putamen Right 1 60 2.4 .times. 10.sup.11 Left 1 60
Right: Caudate Right 2 56.4 + 31 2.8 .times. 10.sup.11 Left 2 33.6
+ 31 AAV2-eGFP 1.71 .times. 10.sup.11 8 Putamen Right 1 62 (PCL)
Left 1 60 Caudate Right 2 30 + 20 Left 1 30 9 Putamen Right 2 20 +
35 Left 2 42 + 19.6 Caudate Right 1 30 Left 1 30
[0206] The first cohort of animals received 1.7.times.10.sup.11 vg
of AAV1-eGFP (TT) (n=3), or AAV2-eGFP (TT) (n=2) per hemisphere.
The second cohort of animals received 1.7.times.10.sup.11 vg of
AAV1-eGFP (PCL) (n=2), or 1.3.times.10.sup.11 vg of AAV2-eGFP (PCL)
(n=2). Serial MRI was acquired to monitor infusate distribution
within each target site and provide real-time feedback to the team.
Immediately after the intraparenchymal dosing procedure, animals
were transferred to the operating room, the guide cannula removed,
and wound site closed in anatomical layers. All experiments were
performed in accordance with National Institutes of Health
guidelines and with protocols approved by the institutional Animal
Care and Use Committee. Immediately after surgery, the animals were
transferred to the MRI suite for AAV dosing procedures. Comments
regarding the dosing procedure for each of the subjects depicted in
Table 2 are provided below.
Dosing Procedure--Treatment Group 2 (AAV1-eGFP TT)
[0207] Subject Number 1.
[0208] Approximately 60 .mu.L of infusate was administered per
hemisphere via two trajectories (304/deposit) into each putamen.
Coronal images from infusions into the anterior putamen showed a
majority of gadolinium signal within each of the targeted
structures. In the right hemisphere, slight perivascular transport
was seen in the ventral putamen and distribution into the anterior
commissure.
[0209] Subject Number 2.
[0210] T1 MRI was acquired after 0.127 mL infusion into the putamen
and 0.207 mL into the thalamus. Post infusion T1 MRI showed
extensive infusate distribution within the right thalamus measuring
approximately 1 cm in the anterior-posterior direction and 1 cm in
the dorso-ventral direction. Coronal T1 showed gadolinium
distribution within the target site that extended medially towards
the internal capsule and superiorly toward the dorsal putamen. A
majority of the infusate was contained within the putaminal
margins; however, transport via the perivascular space was also
present in white matter tracts of the internal capsule and anterior
commissure. Analysis revealed that the ratio of gadolinium infusion
volume (Vi) to distribution volume (Vd) in the thalamus and putamen
was 1:2.
In-Life Observations
[0211] Detailed observations of animal health and neurological
symptoms were performed on a daily basis for a period of 5 days
after dosing; subsequently, detailed observations were performed
once per week until study termination. Observations and daily
mortality checks were performed. Body weights assessments were
performed before intracranial dosing, at time of blood collection
procedures, and at necropsy. No significant difference in body
weight was observed between the treatment groups prior to surgery
or at the time of necropsy (FIG. 1). Whole blood, blood serum and
cerebrospinal fluid (CSF) were collected for hematology, serum
chemistry, AAV1 and AAV2 capsid antibody assay and eGFP mRNA level
analysis.
In-Life Blood Collection and Processing
[0212] Blood (approximately 5 mL) was collected prior to injection,
approximately 72 hours post-injection, and at necropsy according to
the Blood Sample Collection Schedule (see Table 3 below).
Approximately 0.5-1.0 mL of whole blood was collected into EDTA
tubes for hematology analysis. Approximately 2.0 mL of whole blood
was collected into serum separator tubes (with gel, BD Microtainer)
and processed to serum to obtain approximately serum for chemistry
analysis and AAV1 and AAV2 Capsid antibody analysis.
TABLE-US-00003 TABLE 3 Blood sample collection schedule AAV1 and
AAV2 Serum Antibody Time Point Hematology Chemistry Analysis Prior
to Injection X X X 72 Hours Post-Injection X X N/A Necropsy X X X
Volume of Whole Blood 1 mL 2 mL 2 mL Anticoagulant EDTA N/A N/A X =
samples were collected N/A--not applicable
EDTA--Ethylenediaminetetraacetic acid
CSF Collection and Processing
[0213] CSF was collected at two separate time points: prior to
intracranial dosing and at necropsy. CSF collection was performed
under anesthesia by cervical spinal tap with the animal placed in a
prone position. Prior to test article administration, 1-2 mL of CSF
was collected, frozen on dry ice, and stored at .ltoreq.-60.degree.
C. At necropsy (prior to PBS perfusion) 2-4 mL of CSF were
collected, filtered through a 0.8 micron syringe filter into a
labeled collection tube, and transferred in duplicate (1 mL aliquot
for Capsid Antibody analysis and 2-4 mL aliquot for GFP analysis)
into eppendorf tubes, immediately frozen on dry ice and stored at
.ltoreq.-60.degree. C.
Necropsy and Tissue Collection
[0214] All animals were euthanized at approximately 30 days after
the intracranial dosing procedure. Each animal was euthanized using
intravenous administration of sodium pentobarbital. Following
euthanasia and blood and CSF collection, the body was
transcardially perfused with PBS (under RNAse free conditions),
followed by perfusion with PFA. The descending aorta was clamped to
reduce fixation of peripheral tissues. This procedure was used to
collect fixed brain tissue in addition to fresh peripheral tissue
samples for GFP analysis by QPCR. The Tissue Collection Table lists
the tissues that were collected (Table 4). During PBS perfusion
(prior to initiation of 4% PFA perfusion) biopsy samples (0.5-1.0
cm.sup.3) of select tissues (also listed in the Tissue Collection
Table) were collected under RNAse free conditions with disposable
sterile scalpels (a new scalpel for each individual biopsy) into
RNAse free tubes, and stored frozen at .ltoreq.-60.degree. C.
TABLE-US-00004 TABLE 4 Tissue Collection Tissues Collected into PFA
Tissues Collected Frozen (biopsy punch) Brain Heart Spinal Cord
Kidney Liver Lung Spleen Testes Cervical lymph nodes Quadricep
Sciatic Nerve Optic Nerve Eye
Brain and Spinal Cord Processing
[0215] The entire brain was carefully removed from the animal and
photographed along-side a ruler for scale. Once removed from the
skull the brain was placed into a brain matrix and coronally sliced
into 6 mm blocks. Coronal blocks were stored in PFA and processed
for histology. Relevant blocks containing the frontal cortex and
midbrain regions were sectioned into free floating 40 micron
sections. The entire spinal cord was carefully removed from the
animal. Spinal cord segments were stored in PFA and processed for
histology. A representative segment from the cervical, thoracic,
and lumbar region were sectioned into free floating 40 micron
sections.
[0216] After perfusion with PBS-heparin followed by 4% buffered
paraformaldehyde (PFA), the brain from each animal was cut into
6-mm blocks (coronal plane) using disposable blades and monkey
brain matrix. The sequential blocks (11-12 brain slabs per animal)
were placed horizontally on a white board and photographed with
sequentially assigned letters. All brain blocks were then
post-fixed in 4% buffered PFA for 24 hours. The quality of fixation
for each block was inspected visually (no pink color was observed
within blocks). After PFA-postfixation, each brain block was
processed for free-floating sections by rinsing 3.times. in PBS and
immersion in 30% sucrose (cryopreservation) before cutting into
40-.mu.m free-floating sections.
Production of AAV Vectors
[0217] Prior to clinical evaluation, AAV vectors are typically
produced via the standard triple transfection method (TT) in which
HEK293 cells are co-transfected with two or three plasmids encoding
the cis (vector genome) and trans (AAV rep and cap genes;
adenoviral helper genes E2A, E4, and VA) elements required for
vector packaging (Hauck et al., (2009) Mol. Ther. 17:144-152).
Since input of plasmid DNA may be easily and rapidly modified, this
method allows evaluation of vectors based on diverse serotypes and
harboring a variety of expression cassettes. Despite its
flexibility and relatively fast turn-around time, the transfection
method presents a challenge with regard to scalability, which
limits the suitability of this method for large-scale rAAV vector
production for clinical use.
[0218] At the present time, clinical-grade rAAV is generated at
large scale via the helper virus-free transient transfection
method, the recombinant baculovirus or herpes simplex virus-based
production systems, or packaging/producer cell lines (Ayuso et al.,
(2010) Curr. Gene Ther. 10:423-436). Adeno-associated virus
producer cell lines (PCL) are an effective method for large-scale
production of clinical grade AAV vectors. In this system, a single
plasmid containing three components, the vector sequence, the AAV
rep, and cap genes, and a selectable marker gene is stably
transfected into HeLaS3 cells. However, it is desirable to
determine whether AAV vector derived from producer cell lines is
equivalent in potency to vector generated via other methods, for
example, the standard transient transfection method.
[0219] AAV viral vectors were generated for this study using two
different production methods: triple transfection (TT) and producer
cell line (PCL). Recombinant AAV vectors AAV1-GFP (TT) and AAV2-GFP
(TT) were produced by triple transfection (using calcium phosphate)
of human embryonic kidney carcinoma 293 cells (HEK-293) (referenced
in Xiao et al., (1998) Journal of Virology 72:2224-2232). Briefly,
for the production of AAV vectors by transient transfection, HEK293
cells were transfected using polyethyleneimine (PEI) and a 1:1:1
ratio of the three plasmids (ITR vector, AAV2rep/cap2 or
AAV2rep/cap1, and pAd helper plasmid). The ITR vector plasmid
encoded the cDNA for EGFP downstream of the cytomegalovirus
enhancer/chicken beta actin--hybrid promoter (CBA). The pAd helper
used was pHelper (Stratagene/Agilent Technologies, Santa Clara,
Calif.).
[0220] Recombinant AAV vectors AAV1-GFP (PCL) and AAV2-GFP (PCL)
were produced using an AAV producer cell process (referenced in
Thorne et al., (2009) Human Gene Therapy 20:707-714 and Martin et
al., (2013) Human Gene Therapy Methods 24:253-269). Briefly,
product-specific producer cell lines were generated by stable
transfection of Hela-S3 cells (ATCC CCL-2.2) with a plasmid
containing the rep gene from serotype 2 and a capsid gene from
either serotype 1, or 2, the promoter-heterologous nucleic acid
sequence, the vector genome flanked by AAV2 inverted terminal
repeats (ITRs), and a Puromycin resistance gene. The vector genome
harbored the cDNA for EGFP downstream of the cytomegalovirus
enhancer/chicken beta actin-hybrid promoter, CBA. Transfected cells
were grown in the presence of puromycin to isolate stable
integrants. The cell lines generated were screened to select a lead
clone. The product-specific cell clone was subsequently expanded to
a production bioreactor, and infected with a wild type Adenovirus
as helper to initiate AAV production. Virus was harvested 72 hours
post-infection, the adenovirus was inactivated by heat and removed
by anion exchange methods.
[0221] Purification of AAV from both production platforms was
performed as previously described (Qu, G. et al. (2007) J. Virol.
Methods 140:183-192). The resulting titers of all AAV1 and AAV2-GFP
vectors are shown on Table 5. All vectors were prepared in water
containing 180 mM sodium chloride; 10 mM sodium phosphate (5 mM
NaH.sub.2PO.sub.4.2H.sub.2O+5 mM Na.sub.2HPO.sub.4.H.sub.2O); and
0.001% Poloxamer 188 (Lutrol F68), pH 7.5.
TABLE-US-00005 TABLE 5 Study design table and test articles Vector
Dose per No. of Drug hemisphere Group Animals Test Article
concentration (vg) 1 3 ssAAV2/ 1.90 .times. 10.sup.12 1.7 .times.
10.sup.11 1-CBA-GFP (TT) vg/mL 2 2 ssAAV2/ 1.90 .times. 10.sup.12
1.7 .times. 10.sup.11 2-CBA-GFP (TT) vg/mL 3 2 ssAAV2/ 2.30 .times.
10.sup.12 1.7 .times. 10.sup.11 1-CBA-GFP (PCL) vg/mL 4 2 ssAAV2/
1.43 .times. 10.sup.12 1.3 .times. 10.sup.11 2-CBA-GFP (PCL)
vg/mL
Immunohistochemistry
[0222] Immunostaining with antibodies against GFP (1:500, AB3080;
Chemicon) was performed on Zamboni fixed 40-.mu.m coronal sections
covering the entire frontal cortex and extending in a posterior
direction to the level of the striatum. The localization of GFP
immunopositive neurons was analyzed with reference to The Rhesus
Monkey Brain in Stereotactic Coordinates (Paxinos, G. H. X. and
Toga, A. W. (2000) San Diego, Calif.: Academic Press) to identify
specific areas of immunostaining in the cortex and striatum.
[0223] GFP Staining by 3,3'-Diaminobenzidine (DAB):
[0224] Sections (3 per each 6-mm block: separation of 2 mm) were
washed 3 times in PBST for 5 min each followed by treatment with 1%
H.sub.2O.sub.2 for 20 min. Sections were incubated in Sniper
blocking solution (available online at
biocare.net/product/background-sniper/) for 30 min at room
temperature followed by overnight incubation with the primary
anti-GFP antibody (available online at www.lifetechnologies.com/)
diluted 1:1000 in Da Vinci Green Diluent (available online at
biocare.net/). After 3 rinses in PBS containing 0.1% Tween-20
(PBST) for 5 min each, sections were incubated in Mach 2 HRP
polymer (http://biocare.net/) for 1 h, followed by 3 washes and
colorimetric development (DAB). Immunostained sections were
counterstained with cresyl violet and mounted on slides and sealed
with Cytoseal.RTM. (available online at
www.thermoscientific.com/).
[0225] Calculation of Coverage of GFP Expression in the Non-Human
Primate (NHP) Brain:
[0226] GFP staining from matching IHC-stained serial sections was
projected onto individual corresponding MRI scans of each monkey
brain (T1-weighted MR images in the coronal plane).
Distribution/coverage of GFP expression was performed with OsiriX
Imaging Software version 3.1 (The OsiriX Foundation, Geneva,
Switzerland).
[0227] Double-Immunofluorescence for Vector Tropism and Efficiency
of Neuronal Transduction:
[0228] For double fluorescence immunostaining of different cellular
markers (NeuN, S-100, Iba1) with GFP, a combination of primary
antibodies was applied to sections as a cocktail of primary
antibodies by overnight incubation at room temperature in PBST with
20% horse serum. Primary antibodies used were as follows: anti-GFP
antibody (1:500, as above); anti-NeuN (1:500, available online at
www.emdmillipore.com/); anti-S-100 (1:300, available online at
biocare.net/), anti-Iba1 (1:500, available online at biocare.net/);
anti-Olig2 (1:50, available online at www.emdmillipore.com/). After
3 washes in PBST, primary antibodies were visualized by incubation
in the dark for 2 hours with appropriate secondary
fluorochrome-conjugated antibodies: goat anti-mouse DyLight 549 and
goat anti-rabbit DyLight 488 (available online at
www.biocare.net/). All secondary antibodies were diluted 1:1,000 in
Fluorescence Antibody Diluent (available online at biocare.net/).
In addition, to quench autofluorescence, sections were incubated in
0.1% Sudan Black solution (70% ethanol). After final washes in PBS,
sections were cover-slipped with Vectashield Hard Set Mounting
Medium for Fluorescence (available online at www.vectorlabs.com/).
Control sections were processed without primary antibodies, and no
significant immunostaining was observed under these conditions.
[0229] Zeiss Axioskop fluorescence microscope (available online at
www.zeiss.com/) equipped with CCD color video camera and image
analysis system (Axiovision Software, available online at
www.zeiss.com/) was used to determine the presence of
double-labeled cells (positive in both red and green channels).
Photomicrographs for double-labeled sections were obtained by
merging images from two separate channels (red and green;
co-localization appears yellow) without altering the position of
the sections or focus (objective.times.20). For GFP/NeuN
double-staining, 3 sections from each monkey at .about.4-mm
intervals were selected from the sites of vector infusion. For
evaluation of efficiency of neuronal transduction by AAV1-eGFP or
AAV2-eGFP vectors within the targeted brain areas (caudate and
putamen), 5 counting frames (700 .mu.m.times.550 .mu.m) were placed
randomly in the GFP+ area. The primary area of transduction (PAT)
was defined as GFP-positive area ("cloud") that covered more than
40% of the targeted structure. Similarly, to evaluate the
efficiency of neuronal transduction, outside PAT (OPAT), 5 counting
frames (700 .mu.m.times.550 .mu.m) from each section were chosen
beyond the clear margins of GFP-positive "cloud" in the targeted
structures (caudate and putamen) or in the cortex (FIG. 10C). To
determine the proportion of GFP/NeuN-positive cells, each counting
frame was counted twice, first with the red channel for the number
of NeuN+ cells and second with a combined red and green channel for
the number of co-stained cells (GFP+ and NeuN+). At least 1,500
NeuN+ cells were counted for each of the 3 chosen sections (5
counting frames per section). Finally, the percentage of GFP+/NeuN+
to total NeuN+ was determined. All of the calculations for the
striatum were made by adding results from both hemispheres of each
animal and combining values from putamen and caudate nucleus since
the mean transduction efficiencies were identical in both
structures of each animal.
Quantitative Real-Time PCR (TaqMan)
[0230] GFP mRNA levels were measured by quantitative real-time PCR.
Liver, heart, lung, kidney, and spleen samples were used for all
RT-PCR analysis. Total RNA was extracted using the QIAGEN miRNeasy
mini kit and then reverse transcribed and amplified using the High
Capacity cDNA Reverse Transcription Kit (Applied Biosystems)
according to the manufacturer's instructions. Quantitative RT-PCR
reactions were conducted and analyzed on a QuantStudio12K Flex
Real-Time PCR System (Applied Biosystems). Each sample was run in
duplicate and the relative gene expression was determined using a
standard curve.
MR Imaging Data Analysis
[0231] Semi-Quantitative Analyses (Digital MRIs Vd/Vi):
[0232] Distribution volume (Vd) analysis was performed with OsiriX
Imaging Software version 3.1 (The OsiriX Foundation, Geneva,
Switzerland). Infusion sites, cannula tracks and cannula tip were
identified on T1-weighted MR images in the coronal plane.
Regions-of-interest (ROIs) were delineated to outline T1 gadolinium
signals and target sites (i.e. putamen and caudate nucleus).
Three-dimensional volumetric reconstructions of the image series
and ROI were analyzed to estimate volume of distribution (Vd) of
infusions and ratio to volume of infusate (Vi).
[0233] Histological Analysis of Transgene Expression:
[0234] To assess transgene expression, brain sections were
processed for immunohistochemical analysis (IHC). Animals were
deeply anesthetized with sodium pentobarbital (25 mg/kg i.v.) and
euthanized 4 weeks after administration of the vectors. The brains
were removed and sectioned coronally into 6-mm blocks. The blocks
were post-fixed in buffered paraformaldehyde (4%) for 24 h, washed
briefly in PBS and adjusted in a 30% sucrose/PBS solution for
cryopreservation. The formalin-fixed brain blocks were cut into
40-.mu.m coronal sections in a cryostat. Free-floating sections
spanning the entire brain were collected in series and were kept in
antifreeze solution for further IHC analysis.
[0235] Results
[0236] Both AAV1-GFP and AAV2-GFP vectors drove abundant expression
of GFP from transduced neurons as visualized by
immunohistochemistry. After infusion of AAV1 into the caudate and
putamen by CED, extensive GFP immunostaining was detected in the
caudate and putamen (FIGS. 2C&D), as well as the substantia
nigra (FIG. 2D). In addition to the striatum a large number of
cortical regions of the Rhesus monkey brain were also transduced
(FIGS. 2A-D). Cortical GFP expression was most evident in
prefrontal association cortical areas, the premotor cortex, primary
somatosensory cortical areas, and the primary motor cortex, as well
as extensive regions of the occipital cortex (FIGS. 2A-D).
[0237] A large majority of GFP-positive neurons within the cortex
were identified morphologically as pyramidal neurons located in
cortical lamina IV, with axonal projections into the overlying
layers. The density of GFP-positive neurons was particularly high
in the frontal (FIGS. 3A&B) and occipital cortex (FIGS.
3C&D), where large numbers of neurons (FIGS. 3B&D) in
addition to astrocytes (FIGS. 3A&C) were transduced.
Example 2: Widespread GFP Expression after Intrastriatal AAV2
Vector Delivery
[0238] The ability of AAV2 to efficiently target both striatal and
cortical structures in the Rhesus monkey brain when delivered via
convection-enhanced delivery (CED) was evaluated. AAV vectors
containing GFP cDNA under the control of cytomegalovirus
enhancer/chicken beta-actin (CBA) promoter were infused into the
caudate and putamen of 8 adult Rhesus monkeys using CED according
to the methods described in Example 1 above.
[0239] Infusion of AAV2 into the striatum by CED resulted in GFP
expression in the injected regions (caudate and putamen) (FIG. 4C),
substantia nigra (FIG. 4D), and a large number of cortical regions
of the Rhesus monkey brain (FIGS. 4A-D). Expression of GFP in the
striatum of AAV2 injected animals appeared slightly more restricted
and localized when compared to striatal coverage with AAV1 vectors.
The expression of GFP within the NHP striatum was comprehensive but
relatively contained within the gray matter bounds of the targeted
region, with no evidence of significant infusion related leakage or
reflux of the AAV2-GFP vector into adjacent non-targeted areas.
Cortical GFP expression was evident in the same regions seen for
AAV1. Prefrontal association cortical areas, the premotor cortex,
primary somatosensory cortical areas, and the primary motor cortex,
as well as extensive regions of the occipital cortex were well
transduced (FIGS. 4A-D).
Example 3: Comparability of GFP Expression after Intrastriatal AAV1
and AAV2 Vectors Made by Triple Transfection or Producer Cell Line
Process
[0240] To date the majority of preclinical studies utilize AAV
vectors made by Triple Transfection followed by purification using
cesium chloride gradients or column chromatography. Thus, to
evaluate the impact of vector production on biodistribution in
vivo, two methods of vector production Triple Transfection (TT) or
Producer Cell line (PCL), were compared. AAV1 and AAV2 vectors
generated by these two different manufacturing platforms were
administered via CED, and their distribution within the Rhesus
monkey brain was compared.
[0241] Infusion of AAV1-GFP vectors made by triple transfection
yielded equivalent GFP distribution and coverage when compared to
AAV1-GFP vectors made by the producer cell line process. GFP
distribution was comparable between AAV1-GFP (TT) (FIGS. 5C&D)
and AAV1-GFP (PCL) (FIGS. 5A&B) vectors 30 days following
injection into the striatum of Rhesus monkeys.
[0242] Similar results were seen with the AAV2-GFP vectors. GFP
distribution was similar and comparable between AAV2-GFP (TT)
(FIGS. 6C&D) and AAV2-GFP (PCL) (FIGS. 6A&B) injected
brains.
[0243] To measure infusion performance, AAV1-eGFP (TT); AAV2-eGFP
(TT); AAV1-eGFP (PCL); and AAV2-eGFP (PCL) was infused into each
striatum (60 .mu.l into putamen and 30 .mu.l into caudate nucleus),
using 90 .mu.l of each vector mixed with gadolinium contrast agent
(2 mM Prohance; Bracco Diagnostics, Inc.). Magnetic resonance
images (MRI) from each infusion confirmed that positioning of each
cannula was accurate and infusate covered the target area. All
infusions were well contained in the target structure.
Three-dimensional reconstructions of the infusate distribution
generated from gadolinium signal on MR images showed that both
placement and distribution of infusate were very consistent
throughout all the animals. In addition, the ratio (Vd/Vi) between
volume of distribution (Vd) and volume of infusion (Vi) was
calculated for each delivery and data were consistent across all
infusions. Vd was approximately 3-fold larger (range of 2.1-4.6)
than the Vi (Tables 6 and 7).
TABLE-US-00006 TABLE 6 Vector infusion and extent of distribution
within the brain 4 weeks after transduction (mean .+-. st. dev.).
Cortical coverage V.sub.d/V.sub.i .sup.a Gadolinium coverage .sup.b
of GFP expression .sup.c AAV2-eGFP 2.79 .+-. 0.44 Putamen: 29.5
.+-. 10.9% 62.2 .+-. 19.1% Caudate: 18.3 .+-. 5.2% 3.29 .+-. 0.75
Putamen: 23.5 .+-. 9.3% 61.3 .+-. 14.8% Caudate: 24.6 .+-. 8.0%
.sup.a Ratio of volume of distribution (Vd) to volume of infusion
(Vi) was calculated (OsiriX Imaging software, v. 3.1) by dividing
the volume of vector distribution within the injected brain
parenchyma (based on the Gadolinium signal from MRI scans) by the
volume of the injected vector. Values from left and right
hemispheres were added to determine the average Vd/Vi for each
animal. .sup.b Gadolinium coverage within targeted structures was
calculated (OsiriX Imaging software, v. 3.1) by dividing Vd by the
volume of Putamen (600 mm.sup.3) or Caudate (500 mm.sup.3). .sup.c
Cortical GFP coverage was calculated by projecting GFP signal from
matching IHC-stained sections onto corresponding MRI scans of each
monkey (BrainLab software).
TABLE-US-00007 TABLE 7 Vector infusion and extent of distribution
within the brain 4 weeks after transduction (individual values).
Cortical GFP NHP subject V.sub.d/V.sub.i .sup.a Gadolinium coverage
.sup.b coverage .sup.c AAV1-eGFP 1 Putamen: 3.2 Putamen.sub.L 34.8%
Putamen.sub.R 30.2% 91% AAV1-eGFP (TT) Caudate: 2.1 Caudate.sub.L
14.8% Caudate.sub.R 16.2% 2 Putamen: 3.1 Putamen.sub.L 35.7%
Putamen.sub.R 18.3% 50% AAV1-eGFP (TT) Caudate: 3.2 Caudate.sub.L
18.2% Caudate.sub.R 13.8% 3 Putamen: 3.3 Putamen.sub.L 52.7%
Putamen.sub.R 37.8% 41% AAV1-eGFP (TT) Caudate: 2.2 Caudate.sub.L
19.4% Caudate.sub.R 21.2% 4 Putamen: 3.0 Putamen.sub.L 23.3%
Putamen.sub.R 17.7% 61% AAV1-eGFP (PCL) Caudate: 2.3 Caudate.sub.L
23.8% Caudate.sub.R 20.6% 5 Putamen: 2.7 Putamen.sub.L 22.3%
Putamen.sub.R 22.2% 68% AAV1-eGFP (PCL) Caudate: 2.8 Caudate.sub.L
26.8% Caudate.sub.R 8.6% AAV2-eGFP 6 Putamen: 2.7 Putamen.sub.L
22.7% Putamen.sub.R 16.2% 75% AAV2-eGFP (TT) Caudate: 3.7
Caudate.sub.L 20.0% Caudate.sub.R 23.0% 7 Putamen: 2.9
Putamen.sub.L 11.3% Putamen.sub.R 16.3% 47% AAV2-eGFP (TT) Caudate:
3.9 Caudate.sub.L 20.6% Caudate.sub.R 15.2% 8 Putamen: 2.4
Putamen.sub.L 36.0% Putamen.sub.R 19.8% 50% AAV2-eGFP (PCL)
Caudate: 3.4 Caudate.sub.L 34.0% Caudate.sub.R 39.4% 9 Putamen: 4.6
Putamen.sub.L 35.2% Putamen.sub.R 30.5% 73% AAV2-eGFP (PCL)
Caudate: 2.7 Caudate.sub.L 21.2% Caudate.sub.R 23.4% .sup.a Ratio
of volume of distribution (Vd) to volume of infusion (Vd) was
calculated (OsiriX Imaging software, v. 3.1) by dividing the volume
of vector distribution within the injected brain parenchyma (based
on the Gadolinium signal from MRI scans) by the volume of the
injected vector. Values from left and right hemispheres were added
to determine the average Vd/Vi for each animal. .sup.b Gadolinium
coverage within targeted structures was calculated (OsiriX Imaging
software, v. 3.1) by dividing Vd by the volume of Putamen (600
mm.sup.3) or Caudate (500 mm.sup.3). .sup.c Cortical GFP coverage
was calculated by projecting GFP signal from matching IHC-stained
sections onto corresponding MRI scans of each monkey.
[0244] After bilateral injection of both AAV1-eGFP and AAV2-eGFP
(prepared by both methods of productions, TT and PCL) into the
striatum of NHP, robust expression of eGFP was evident throughout
both the target structures (putamen and caudate nucleus) as well as
projection regions (external and internal globus pallidus--GPe and
GPi, substantia nigra--SN, subthalamic nucleus--STN, cortical
regions--neuronal layers IV and V) regions (FIG. 7).
[0245] To evaluate role of Gadolinium (Gd) as a marker of vector
distribution, the ratio of the area of GFP expression (from
histological sections) to the area of Gadolinium signal on
corresponding MR scans was calculated. For monkeys infused with
serotype AAV1, this ratio was 1.21.+-.0.10 whereas for AAV2 it was
0.74.+-.0.04 (FIG. 8). The ratio of 1.0 indicates a perfect match
between GFP expression and vector distribution as determined by
MRI. This difference indicated that AAV1 vector distributed beyond
the Gd signal and achieved better spread in the primary area of
transduction than AAV2.
[0246] To evaluate the distribution of the infused AAV vectors
within the brain, representative free-floating brain sections (3
per each block; 40-.mu.m thick) from each animal were stained with
a rabbit anti-GFP antibody (Millipore; Cat. No. 3850, dilution
1:500). Immunohistochemical assessment revealed dark brown DAB
signal within the injected targets (right and left putamens and
caudates) as well as multiple areas projected from the injected
areas (cortical regions).
[0247] By projecting the extent of GFP expression onto MRI scans of
each monkey brain, the percentage of coverage for cortical regions
(brain areas relevant in HD) was calculated (see Table 6 for
summary and Table 7 for further details). In all monkeys, 24% of
striatum was transduced on average, which resulted in substantial
expression of GFP in the cortex (FIG. 7). The extent of GFP
expression in the cortex did not correlate with the AAV serotype
used (AAV1 vs. AAV2) or the method of vector production (TT vs.
PCL). It seems that broader distribution of infusate within the
infusion site (striatum) was a key driver of the extent of
transduction in the cortex. One NHP (Subject No. 1; AAV1-eGFP [TT])
showed a particularly robust spread of GFP expression into cortical
regions (layer IV and V) of the entire brain (both frontal and
occipital--see FIG. 7). Other animals showed variability in
cortical expression associated with variations both in the extent
and in localized anatomical regions within caudate and putamen.
Since pre- and commissural regions of the striatum were targeted,
GFP was detected more in frontal and parietal cortical regions and
less in the occipital cortex. Histological analysis for each animal
is summarized below (grouped by treatment group).
[0248] Treatment Group 2 (ssAAV2/1-CBA-eGFP TT)
[0249] Immunohistochemical evaluation of eGFP expression in the
whole brain revealed robust signal in the targeted sites (both
putamens and caudate nuclei) and projected structures (globus
pallidus, substantia nigra, thalamus, subthalamic nucleus, and
cortical regions).
[0250] Subject Number 1.
[0251] Subject showed a particularly robust spread of GFP
expression to cortical regions (layer 4 and 5) of the entire brain
(both frontal and occipital). The morphology of the GFP-positive
cells implied both neuronal and astrocytic transduction, which was
later confirmed by double immunofluorescence staining (see below).
The calculation of GFP expression coverage showed that 91% of the
entire cortex (see Table 7) was transduced (this calculation was
done by projecting the extent of GFP signal onto MRI scans of the
analyzed monkey brain).
[0252] Subject Number 2.
[0253] Subject showed robust transduction in putamens and caudate
nuclei as well as globus pallidus and substantia nigra of both
hemispheres. The projection of GFP expression to cortex was less
pronounced than in subject number 1 and was observed mainly in the
frontal regions of the cortex. Although GFP signal was also
detected in occipital cortex, the density of GFP-positive cells was
significantly lower. The cortical GFP expression coverage was
accounted for 50% (Table 7). Similarly as in subject number 1,
GFP-positive cells had both neuronal and astrocytic morphology,
which was confirmed by double immunofluorescence.
[0254] Subject Number 3.
[0255] Subject showed strong GFP expression in putamens and caudate
nuclei as well as all projected structures (globus pallidus,
substantia nigra, subthalamic nucleus, thalamus, and cortex).
Although GFP signal was clearly detected in some regions of cortex,
GFP expression was accounted for only 41% of its overall cortical
coverage (the lowest in all tested monkeys; see Table 7). Both
neurons and astrocytes were transduced. A large part of the right
anterior corona radiate also showed GFP-positive signal, most
likely as a result of vector spillage from the cannula penetrating
to the striatum.
[0256] Treatment Group 3 (ssAAV2/2-CBA-eGFP TT)
[0257] Subject Number 6.
[0258] Subject showed a strong GFP signal in both targeted
structures (putamen and caudate nucleus). Densely scattered
positive cells were detected in both of those regions. The GFP
expression spread also to frontal cortical regions, globus
pallidus, substantia nigra, subthalamic nucleus, and some parts of
thalamus. The GFP expression coverage in the cortex accounted for
75% (Table 7). GFP-positive cells had mostly neuronal morphology,
which was later confirmed by double immunofluorescence.
GFP-positive cells of astrocyte shape were detected in the internal
capsule as well as in a few cortical spots and closely neighboring
white matter areas with clearly visible tracks of the infusion
cannulas.
[0259] Subject Number 7.
[0260] Subject showed GFP-positive signal within the right and left
striatum (both putamen and caudate nucleus). Its distribution was
rather poor and pattern appeared "spotty" rather than uniform when
compared to other infused monkeys. Consequently, the GFP signal in
all projected brain structures appeared weaker as well. The GFP
expression coverage in the cortex accounted for 47% (Table 7).
GFP-positive cells had mostly neuronal morphology, which was later
confirmed by double immunofluorescence. GFP-positive cells of
astrocyte shape were detected in the internal capsule as well as in
a few cortical spots and closely neighboring white matter areas
with clearly visible tracks of the infusion cannulas.
[0261] Treatment Group 4 (ssAAV2/1-CBA-eGFP PCL)
[0262] Subject Number 4.
[0263] Subject showed very robust GFP expression in targeted
structures, putamen and caudate nucleus. GFP-positive signal was
also detected in globus pallidus, substantia nigra, subthalamic
nucleus, thalamus and cortical regions. The GFP expression coverage
in the cortex accounted for 61% (Table 7). Anterior part of the
corona radiata also showed GFP-positive signal, mostly likely as a
result of vector spillage from the cannulas penetrating to the
striatum. Positive cells had both neuronal and astrocytic
morphology, which was later confirmed by double
immunofluorescence.
[0264] Subject Number 5.
[0265] Subject showed robust GFP expression in the striatum (both
putamen and caudate nucleus). GFP-signal was also detected in
projected structures (globus pallidus, substantia nigra,
subthalamic nucleus, thalamus and cortical regions). The GFP
expression coverage in the cortex accounted for 68% (Table 7).
Anterior part of the corona radiata also showed GFP-positive
signal, mostly likely as a result of vector spillage from the
cannulas penetrating to the striatum. Positive cells had both
neuronal and astrocytic morphology.
[0266] Treatment Group 5 (ssAAV2/2-CBA-eGFP PCL)
[0267] Subject Number 9.
[0268] Subject showed very robust GFP expression in the striatum
(both putamen and caudate nucleus). GFP signal was also detected in
projected structures (globus pallidus, substantia nigra,
subthalamic nucleus, thalamus and cortical regions). The GFP
expression coverage in the cortex accounted for 73% (Table 7).
GFP-positive cells had mostly neuronal morphology although
GFP-positive cells of astrocyte shape were also detected within
white matter tracts (internal capsule) and immediate vicinity of
cannula tracks.
[0269] Subject Number 8.
[0270] Subject showed robust GFP expression in the striatum and
projected structures (globus pallidus, substantia nigra,
subthalamic nucleus, thalamus and cortical regions). The GFP
expression coverage in the cortex accounted for 50% (Table 7).
GFP-positive cells had mostly neuronal morphology although GFP+
cells of astrocyte shape were also detected within white matter
tracts (internal capsule, corona radiata) and in the immediate
vicinity of cannula tracks.
Double Immunofluorescence
[0271] For both groups of NHPs transduced with AAV1-eGFP vectors
(TT and PCL), the morphology of GFP-positive cells suggested both
neuronal and astrocytic transduction (FIGS. 9A-9D). This was
confirmed by double immunofluorescence staining with a combination
of antibodies against GFP and NeuN (neuronal marker) or GFP and
S-100 (astrocytic marker) (FIGS. 10A-10C). In contrast, AAV2-eGFP
(both TT and PCL) directed predominantly neuronal transduction
(FIGS. 9E-9G and 10D). GFP-positive cells of astrocytic lineage
were also detected in the internal capsule (FIG. 9H) as well as in
cortical regions of white matter where the infusion cannula tracks
were visible.
[0272] Based on double immunofluorescence, the efficiency of
neuronal transduction in the striatum and cortical regions was
calculated (at the coronal plane of the infusion site) for all
NHPs. FIG. 11A summarizes the findings in the striatum. Individual
calculations for each animal are shown in Table 8 below.
TABLE-US-00008 TABLE 8 Efficiency of neuronal transduction by
AAV1-eGFP and AAV2-eGFP vectors within the striatal primary areas
of transduction (PAT) and the cortex of the non-human primate
brain. Subject No. 1 2 3 6 7 AAV1-eGFP AAV1-eGFP AAV1-eGFP
AAV2-eGFP AAV2-eGFP Targeted region (TT) (TT) (TT) (TT) (TT) Left
putamen 57.0 .+-. 7.75% 64.4 .+-. 11.75% 54.7 .+-. 11.4% 36.5 .+-.
8.8% 59.6 .+-. 12.8% Right putamen 68.0 .+-. 15.9% 70.1 .+-. 14.4%
66.2 .+-. 20.0% 33.1 .+-. 11.7% 56.3 .+-. 7.7% Left caudate 70.1
.+-. 7.4% 72.6 .+-. 13.3% 56.4 .+-. 6.02% 33.7 .+-. 11.4% 61.4 .+-.
13.1% Right caudate 65.6 .+-. 7.75% 65.1 .+-. 13.9% 60.1 .+-. 5.65%
42.7 .+-. 10.5% 49.4 .+-. 13.5% Cortex* 24.8 .+-. 3.04% 4.04 .+-.
2.77% 6.75 .+-. 3.38% 8.56 .+-. 2.81% 3.36 .+-. 1.83% Subject No. 5
4 9 8 AAV1-eGFP AAV1-eGFP AAV2-eGFP AAV2-eGFP Targeted region (PCL)
(PCL) (PCL) (PCL) Left putamen 58.6 .+-. 7.61% 57.1 .+-. 10.5% 53.1
.+-. 12.7% 52.4 .+-. 11.8% Right putamen 50.5 .+-. 9.78% 73.2 .+-.
9.88% 52.1 .+-. 10.6% 50.1 .+-. 4.5% Left caudate 57.0 .+-. 7.13%
51.8 .+-. 9.42% 43.0 .+-. 14.5% 43.4 .+-. 8.5% Right caudate 59.1
.+-. 9.11% 70.5 .+-. 8.38% 52.1 .+-. 7.22% 61.2 .+-. 12.9% Cortex*
16.3 .+-. 5.09% 16.1 .+-. 6.59% 23.0 .+-. 7.55% 18.6 .+-. 5.36%
*Neuronal transduction by AAV vectors was also detected in cortical
regions projected from the striatum (target structure). The
efficiency of cortical transduction was calculated in coronal
sections of the striatal plane with injection sites.
[0273] Striatal neuronal transduction in the regions of primary
transduction, indicated by MRI, ranged from 50% to 65%. The highest
efficiency of transduction was observed in NHPs infused with
AAV1-eGFP (TT) with the mean of 64.2.+-.5.9% and the lowest in
group AAV2-eGFP (TT) with the mean of 46.6.+-.11.7% (p<0.05;
2-way ANOVA). This suggests that serotype AAV1 has .about.18%
higher efficiency in transducing neurons than AAV2. AAV1-eGFP
produced by PCL evinced a weaker trend (p>0.05; 2-way ANOVA)
toward transducing more neurons (59.7.+-.8.1%) than AAV2-eGFP
(50.1.+-.5.8%).
[0274] The above calculations were derived from areas of strong GFP
transduction as defined by MRI, (primary area of
transduction--PAT). In addition, the efficiency of neuronal
striatal transduction in regions outside the PAT was calculated to
see if GFP-positive cells could also be detected outside the clear
boundary of strong GFP signal ("outside the primary area of
transduction"--OPAT), suggesting perhaps that all tested vectors
spread in the same manner. The scheme of how these areas were
chosen (random selection of 5 counting frames) is illustrated in
FIG. 11B. A dramatic difference in the estimation of transduction
efficiency in OPAT between serotypes AAV1 and AAV2 was observed
(FIG. 11C), with AAV1 transducing many more neurons than AAV2
(8.1.+-.3.8% vs. 0.74.+-.0.25% for TT groups and 7.2.+-.3.5% vs.
2.16.+-.1.8% for PCL groups; p<0.05 in both comparisons 2-way
ANOVA;). Individual calculations for each animal are shown in Table
9 below.
TABLE-US-00009 TABLE 9 Efficiency of neuronal transduction by
AAV1-eGFP and AAV2-eGFP vectors within the striatum but outside the
primary areas of transduction (OPAT) of the non-human primate
brain. Subject No. 1 2 3 6 7 AAV1-eGFP AAV1-eGFP AAV1-eGFP
AAV2-eGFP AAV2-eGFP Targeted region (TT) (TT) (TT) (TT) (TT) Left
putamen 14.2 .+-. 9.49% 6.88 .+-. 6.48% 14.2 .+-. 7.87% 0.57 .+-.
0.53% 1.28 .+-. 0.69% Right putamen 4.36 .+-. 2.06% 4.04 .+-. 4.36%
5.35 .+-. 3.68% 0.51 .+-. 0.61% 0.80 .+-. 0.55% Left caudate 10.1
.+-. 7.42% 2.01 .+-. 2.54% 9.11 .+-. 5.24% 0.86 .+-. 0.95% 0.71
.+-. 0.36% Right caudate 10.8 .+-. 6.37% 6.89 .+-. 5.62% 8.76 .+-.
3.50% 0.51 .+-. 0.91% 0.71 .+-. 0.93% Subject No. 5 4 9 8 AAV1-eGFP
AAV1-eGFP AAV2-eGFP AAV2-eGFP Targeted region (PCL) (PCL) (PCL)
(PCL) Left putamen 2.82 .+-. 1.99% 12.3 .+-. 6.27% 1.16 .+-. 0.87%
1.98 .+-. 2.20% Right putamen 3.59 .+-. 3.01% 9.83 .+-. 5.64% 1.17
.+-. 1.18% 2.68 .+-. 0.90% Left caudate 7.11 .+-. 4.61% 10.7 .+-.
5.53% 0.85 .+-. 0.63% 3.61 .+-. 3.22% Right caudate 6.95 .+-. 5.82%
4.18 .+-. 2.94% 1.13 .+-. 0.65% 1.99 .+-. 1.26%
[0275] In addition, the efficiency of transduction in cortical
regions projecting to the striatum was calculated. Since the degree
of cortical coverage varied among animals, random cortical areas
were counted in sections with adjacent GFP-positive striatum. There
was an evident discrepancy observed among the animals (Table 8)
with no clear correlation with the serotype used. The mean neuronal
transduction efficiency for AAV1 was 13.6.+-.8.3% vs. 13.4.+-.9.0%
for AAV2 (p>0.97).
[0276] As mentioned above, AAV1-eGFP transduced many more
astrocytes (S-100 marker) than neurons. GFP-positive astrocytes
were detected in the sites of primary transduction (striatum) as
well as in cortical regions projecting to striatum. Examples of
GFP-transduced astrocytes are shown in FIG. 10C. For AAV2-eGFP
vectors, only sporadic GFP+astrocytes could be detected surrounding
the track of the infusion cannulas. To determine whether the
vectors transduced other antigen-presenting cells in the brain,
representative brain sections from all monkeys were co-stained with
antibodies against GFP and Iba-1, specific for microglia. None of
the animals showed double-labeled cells, excluding this possibility
(FIG. 10E). In turn, in all tested monkeys, staining against
Olig-2, the marker for oligodendrocytes, showed only a few cells
positive for both GFP and Olig-2. Those sparse cells were detected
mainly in the vicinity of the cannula tracks (data not shown).
[0277] Brain sections were stained with hematoxylin-eosin (H&E)
to determine whether these vectors triggered neuroinflammation.
Sections were examined mainly for the presence of perivascular
cuffing--the accumulation of lymphocytes or plasma cells in a dense
mass around blood vessels. Although varying degrees of such
infiltrates were detected in all monkeys, no other
vector/transgene-related histological findings were observed.
However, AAV1 was observed to cause slightly more pronounced
infiltration of macrophages and lymphocytes within the primary
areas of transduction than did AAV2 (FIGS. 12A and 12B). Also,
vectors produced by Triple Transfection seemed to cause more
extensive perivascular cuffing than vectors generated by Producer
Cell Line process. Of note, no infiltrates were detected in
projecting areas of transduction (cortical regions).
Example 4: Absence of Detectable GFP Expression in Peripheral
Tissue Outside of the Central Nervous System (CNS)
[0278] Peripheral organ tissues, including kidney, liver, lung,
heart, and spleen, were collected at necropsy to evaluate whether
CED administered AAV-GFP transgene expression could be detected
outside the CNS.
[0279] Results
[0280] No detectable levels of GFP were detected in any of the
organs collected. Both AAV-GFP (TT) (FIG. 13A) and AAV-GFP (PCL)
(FIG. 13B) vectors showed no detectable peripheral expression of
GFP. Mouse brain tissue injected with AAV2/1-GFP (TT) vectors were
used as positive controls for this assay.
CONCLUSIONS
[0281] Efficient delivery of therapeutic proteins to the brain
remains a serious obstacle to achieving clinical efficacy while
minimizing adverse effects. Developments in gene delivery have
provided an opportunity to establish production of biologics within
the brain parenchyma. These advances have led to the initiation of
multiple clinical trials in which AAV vectors have become a
preferred vector system for treating neurologic disorders. Although
focal targeting of a specific nucleus can be reliably accomplished
by stereotactic neurosurgical infusion, the extensive convoluted
arrangement of the human cortex is not easily targeted by direct
infusion of viral vectors. The difficulties in safely achieving
widespread gene expression in the brain have hindered the
development of potential treatments for neurologic diseases which
require cortical delivery.
[0282] As described herein, AAV vectors (e.g., AAV1 and AAV2) are
capable of providing extensive delivery to the entire primate
striatum (caudate and putamen), as well as delivering to
significant numbers of cells within the cerebral cortex (including
frontal cortex, occipital cortex, and layer IV), thalamus, and
hippocampus. GFP, a reporter protein with no known function in the
cerebral cortex, was utilized in the studies discussed herein. AAV1
and AAV2-GFP infused into the caudate and putamen using a CED
delivery method resulted in a high level of GFP expression in both
caudate and putamen as well as several regions of the cortex.
GFP-positive neurons in the frontal cortex were located >20 mm
from the AAV-GFP infusion site, thereby demonstrating axonal
transportation of the GFP protein and AAV vector. Without wishing
to be bound by theory, because GFP remains cytoplasmic and is not a
secreted protein, the presence of GFP in the cortex is thought to
indicate direct cellular transduction and active transportation of
AAV2 vector along single axonal projections. Huntington's disease
is an exemplary disease for which striatal delivery of AAVs (e.g.,
CED striatal delivery) may be useful. Huntington's disease affects
both striatal and cortical regions and thus a therapeutic strategy
that targets both areas is ideal.
[0283] The findings disclosed herein underscore the potential for
delivery of AAV vectors (e.g., AAV1 and AAV2) to transduce neurons
located a considerable distance from the striatal infusion site.
Intrastriatal administration of AAV vectors (e.g., AAV1 and AAV2)
is therefore ideal for use in treating CNS disorders that require
delivery of therapeutic molecules to the striatum and cortex,
including but not limited to, Huntington's disease. In addition, as
AAV vectors generated by the triple transfection method and
producer cell line method show comparable transgene expression
patterns and levels of transduction, triple transfection and
producer cell line methods of generating AAV vectors are suitable
for use in the present invention.
Sequence CWU 1
1
1178DNAArtificial SequenceSynthetic Construct 1cactccctct
ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccacgc 60ccgggctttg
cccgggcg 78
* * * * *
References